Plasmid maintenance system for antigen delivery

ABSTRACT

The present invention relates generally to a Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens, and methods for making and using the Plasmid Maintenance System. The invention optimizes the maintenance of expression plasmids at two independent levels by: (1) removing sole dependence on balanced lethal maintenance functions; and (2) incorporating at least one plasmid partition function to prevent random segregation of expression plasmids, thereby enhancing their inheritance and stability. The Plasmid Maintenance System may be employed within a plasmid which has been recombinantly engineered to express a variety of expression products.

BACKGROUND OF THE INVENTION

[0001] 1.1 Field of the Invention

[0002] The present invention relates generally to expression plasmidsstabilized by a Plasmid Maintenance System (as defined herein) capableof expressing a protein or peptide, such as an antigen for use in a livevector vaccine, and methods for making and using the stabilizedplasmids. The invention optimizes the maintenance of expression plasmidsat two independent levels by: (1) removing sole dependence on catalyticbalanced lethal maintenance systems; and (2) incorporating a plasmidpartition system to prevent random segregation of expression plasmids,thereby enhancing inheritance and stability.

[0003] 1.2 Description of Related Art Set forth below is a discussion ofart relevant to the present invention.

[0004] 1.2.1 Bacterial Live Vector Vaccines

[0005] Bacterial live vector vaccines deliver antigens to a host immunesystem by expressing the antigens from genetic material contained withina bacterial live vector. The genetic material is typically a replicon,such as a plasmid. The antigens may include a wide variety of proteinsand/or peptides of bacterial, viral, parasitic or other origin.

[0006] Among the bacterial live vectors currently under investigationare attenuated enteric pathogens (e.g., Salmonella typhi, Shigella,Vibrio cholerae), commensals (e.g., Lactobacillus, Streptococcusgordonil) and licensed vaccine strains (e.g., BCG). S. typhi is aparticularly attractive strain for human vaccination.

[0007] 1.2.2 Attenuated Salmonella typhi as a Live Vector Strain

[0008]S. typhi is a well-tolerated live vector that can deliver multipleunrelated immunogenic antigens to the human immune system. S. typhi livevectors have been shown to elicit antibodies and a cellular immuneresponse to an expressed antigen. Examples of antigens successfullydelivered by S. typhi include the non-toxigenic yet highly immunogenicfragment C of tetanus toxin and the malaria circumsporozoite proteinfrom Plasmodium falciparum.

[0009]S. typhi is characterized by enteric routes of infection, aquality which permits oral vaccine delivery. S. typhi also infectsmonocytes and macrophages and can therefore target antigens toprofessional APCs.

[0010] Expression of an antigen by S. typhi generally requiresincorporation of a recombinant plasmid encoding the antigen.Consequently, plasmid stability is a key factor in the development ofhigh quality attenuated S. typhi vaccines with the ability toconsistently express foreign antigens.

[0011] Attenuated S. typhi vaccine candidates for use in humans shouldpossess at least two well separated and well defined mutations thatindependently cause attenuation, since the chance of in vivo reversionof such double mutants would be negligible. The attenuated vaccinecandidate S. typhi CVD908 possesses such properties. CVD908 contains twonon-reverting deletion mutations within the aroC and aroD genes. Thesetwo genes encode enzymes critical in the biosynthetic pathway leading tosynthesis of chorismate, the key precursor required for synthesis of thearomatic amino acids phenylalanine, tyrosine, and tryptophan. Chorismateis also required for the synthesis of p-aminobenzoic acid; after itsconversion to tetrahydrofolate, p-aminobenzoic acid is converted to thepurine nucleotides ATP and GTP.

[0012] 1.2.3 Plasmid Instability

[0013] Plasmidless bacterial cells tend to accumulate more rapidly thanplasmid-bearing cells. One reason for this increased rate ofaccumulation is that the transcription and translation of plasmid genesimposes a metabolic burden which slows cell growth and gives plasmidlesscells a competitive advantage. Furthermore, foreign plasmid geneproducts are sometimes toxic to the host cell.

[0014] Stable inheritance of plasmids is desirable in the field ofattenuated bacterial live vector vaccines to ensure successful continuedantigen production, as well as in commercial bioreactor operations inorder to prevent bioreactor takeover by plasmidless cells.

[0015] Stable inheritance of a plasmid generally requires that: (1) theplasmid must replicate once each generation, (2) copy number deviationsmust be rapidly corrected before cell division, and (3) upon celldivision, the products of plasmid replication must be distributed toboth daughter cells.

[0016] Although chromosomal integration of foreign genes increases thestability of such sequences, the genetic manipulations involved can bedifficult, and the drop in copy number of the heterologous gene oftenresults in production of insufficient levels of heterologous antigen toensure an optimal immune response. Introduction of heterologous genesonto multicopy plasmids maintained within a live vector strain is anatural solution to the copy number problem; genetic manipulation ofsuch plasmids for controlled expression of such heterologous genes isstraightforward. However, resulting plasmids can become unstable invivo, resulting in loss of these foreign genes.

[0017] 1.2.4 Plasmid Stabilization Systems

[0018] In nature bacterial plasmids are often stably maintained, eventhough usually present at very low copy numbers. Stable inheritance ofnaturally occurring lower copy number plasmids can depend on thepresence of certain genetic systems which actively prevent theappearance of plasmid-free progeny. A recent review of plasmidmaintenance systems can be found in Jensen et al. Molecular Microbiol.17:205-210, 1995 (incorporated herein by reference).

[0019] 1.2.5 Antibiotic Resistance

[0020] One means for maintaining plasmids is to provide an antibioticresistance gene on the plasmid and to grow the cells inantibiotic-enriched media. However, this method is subject to a numberof difficulties. The antibiotic resistance approach is expensive,requiring the use of costly antibiotics and, perhaps more importantly,the use of antibiotics in conjunction with in vivo administration ofvaccine vectors is currently discouraged by the U.S. Food and DrugAdministration.

[0021] In large-scale production applications, the use of antibioticsmay impose other limitations. With respect to commercial bioreactors,antibiotic resistance mechanisms can degrade the antibiotic and permit asubstantial population of plasmidless cells to persist in the culture.Such plasmidless cells are unproductive and decrease the output of thebioreactor.

[0022] There is therefore a need in the art for a plasmid maintenancesystem specifically designed for use in bacterial live vector vaccineswhich does not rely on antibiotic resistance, and preferably which isalso useful in commercial bioreactor applications.

[0023] 1.2.6 Segregational Plasmid Maintenance Functions

[0024] Stable lower copy number plasmids typically employ a partitioningfunction that actively distributes plasmid copies between daughtercells. Exemplary partitioning functions include, without limitation,systems of pSC101, the F factor, the P1 prophage, and IncFII drugresistance plasmids. Such functions are referred to herein as “SEG”functions.

[0025] 1.2.7 Post-Segregational Killing (PSK) Functions

[0026] Naturally occurring PSK plasmid maintenance functions typicallyemploy a two component toxin-antitoxin system and generally operate asfollows: The plasmid encodes both a toxin and an antitoxin. Theantitoxins are less stable than the toxins, which tend to be quitestable. In a plasmidless daughter cell, the toxins and anti-toxins areno longer being produced; however, the less stable antitoxins quicklydegrade, thereby freeing the toxin to kill the cell.

[0027] The toxins are generally small proteins and the antitoxins areeither small proteins (proteic systems such as phd-doc) or antisenseRNAs which bind to the toxin-encoding mRNAs preventing their synthesis(antisense systems such as hok-sok).

[0028] Balanced lethal systems discussed below in Section 1.2.7.3 are anexample of an artificial PSK function.

[0029] 1.2.7.1 Proteic Maintenance System: The phd-doc System

[0030] In proteic PSK functions, both the toxin and antitoxin aresynthesized from operons in which the gene encoding the antitoxin isupstream of the gene encoding the toxin. These operons autoregulatetranscription levels, and synthesis of the encoded proteins istranslationally coupled. The antitoxin is generally synthesized inexcess to ensure that toxin action is blocked. The unstable antitoxinsare constantly degraded by host-encoded proteases, requiring constantsynthesis of antitoxin to protect the cell. Upon loss of the plasmid,antitoxins are no longer produced, and the existing antitoxins rapidlydegrade, permitting the toxin to kill the host cell.

[0031] The phd-doc system is an example of a proteic PSK function. Thephd-doc system occurs naturally within the temperate bacteriophage P1,which lysogenizes Escherichia coli, as an ˜100 kb plasmid. Thismaintenance locus encodes two small proteins: the toxic 126 amino acidDoc protein causes death on curing of the plasmid by an unknownmechanism, and the 73 amino acid Phd antitoxin prevents host death,presumably by binding to and blocking the action of Doc.

[0032] Phd and Doc are encoded by a single transcript in which the ATGstart codon of the downstream doc gene overlaps by one base the TGA stopcodon of the upstream phd gene. Expression of these two proteins istherefore translationally coupled, with Phd synthesis exceedingsynthesis of the toxic Doc protein.

[0033] In addition, transcription of this operon is autoregulated at thelevel of transcription through the binding of a Phd-Doc protein complexto a site which blocks access of RNA polymerase to the promoter of theoperon as concentrations of both proteins reach a critical level.Although Doc appears to be relatively resistant to proteolytic attack,Phd is highly susceptible to cleavage. The PSK mechanism of aplasmid-encoded phd-doc locus is therefore activated when bacteriaspontaneously lose this resident plasmid, leading to degradation of thePhd antitoxin and subsequent activation of the Doc toxin which causescell death.

[0034] 1.2.7.2 Antisense Maintenance System: The hok-sok System

[0035] In antisense maintenance systems, the antitoxins are antisenseRNAs that inhibit translation of toxin-encoding mRNAs. Like theantitoxin peptides, the antisense RNAs are less stable than thetoxin-encoding mRNA. Loss of the plasmid permits existing antitoxins todegrade, thereby permitting synthesis of the toxin which kills the hostcell.

[0036] An example of an antisense maintenance system is the hok-soksystem, encoded by the parB locus of plasmid R1. The system is comprisedof three genes: hok, sok and mok.

[0037] Hok is a membrane-associated protein which irreversibly damagesthe cell membrane, killing host cells. Expression of Hok from hok mRNAleads to a loss of cell membrane potential, arrest of respiration,changes in cell morphology, and cell death.

[0038] The sok gene encodes a trans-acting RNA which blocks translationof hok mRNA, thereby preventing Hok killing of host cells. The sok RNAis less stable than hok mRNA and is expressed from a relatively weakpromoter. (Gerdes et al. Annu. Rev. Genet., 31:1-31, 1997) incorporatedherein. The mechanism by which sok RNA blocks translation of Hok inplasmid-containing cells became apparent only after the identificationof mok (modulation of killing), a third gene in the parB locus. The mokopen reading frame overlaps with hok, and is necessary for expressionand regulation of hok translation.

[0039] The sok antisense RNA forms a duplex with the 5′ end of themok-hok message rendering the mok ribosome binding site inaccessible toribosomes and promoting RNase III cleavage and degradation of the mRNA.In the absence of mok translation, hok is not expressed from intactmessage, even though its own ribosome binding site is not directlyobscured by sok RNA.

[0040] When a plasmid-free cell is formed, the unstable sok RNA decaysmuch more rapidly than the stable mok-hok message. When the protectionafforded by sok is lost, Mok and Hok are translated and the cell dies.

[0041] A limitation of the hok-sok system is that a significant numberof plasmidless cells can arise when the hok-sok system is inactivated bymutations within the Hok open reading frame.

[0042] 1.2.7.3 Balanced Lethal Systems

[0043] In a balanced-lethal system (a PSK function), a chromosomal geneencoding an essential structural protein or enzyme is deleted from thebacterial chromosome or is mutated such that the gene can no longeroperate. The removed or damaged gene is then replaced by a plasmidcomprising a fully operating gene. Loss of the plasmid results in aninsufficiency of the essential protein and the death of the plasmidlesscell.

[0044] A balanced-lethal system has been successfully employed in S.typhimurium based on expression of the asd gene encoding aspartateβ-semialdehyde dehydrogenase (Asd). Asd is a critical enzyme involved inthe synthesis of L-aspartic-β-semialdehyde, which is a precursoressential for the synthesis of the amino acids L-threonine (andL-isoleucine), L-methionine, and L-lysine, as well as diaminopimelicacid, a key structural component essential to the formation of the cellwall in Gram-negative bacteria. Loss of plasmids encoding Asd would belethal for any bacterium incapable of synthesizing Asd from thechromosome, and would result in lysis of the bacterium due to aninability to correctly assemble the peptidoglycan layer of its cellwall.

[0045] The asd system (a PSK function) has been successfully employed inattenuated S. typhimurium-based live vector strains for immunization ofmice with a variety of procaryotic and eucaryotic antigens, includingsuch diverse antigens as detoxified tetanus toxin fragment C and the LTenterotoxin, synthetic hepatitis B viral peptides, and gamete-specificantigens such as the human sperm antigen SP10.

[0046] Murine mucosal immunization with these live vector strains haselicited significant immune responses involving serum IgG and secretoryIgA responses at mucosal surfaces.

[0047] The asd system has recently been introduced into attenuatedSalmonella typhi vaccine strains in an attempt to increase the stabilityof plasmids expressing synthetic hepatitis B viral peptides. However,when volunteers were immunized with these live vector strains, no immuneresponse to the foreign antigen was detected.

[0048] In fact, to date, very few reports have documented an immuneresponse to plasmid-based expression of a foreign antigen fromstabilized plasmids after human vaccination with an attenuated S. typhilive vector. In one report, the vaccine strain Ty21a was madeauxotrophic for thymine by selecting in the presence of trimethoprim foran undefined mutation in the thyA gene, encoding thymidylate synthetase.

[0049] Although in some cases failure of live vector strains may haveresulted from over-attenuation of the strain itself, it appears probablethat current killing systems for plasmids suffer from additionallimitations. In those situations where the chromosomal copy of the genehas been inactivated, rather than removed, may allow for restoration ofthe chromosomal copy via homologous recombination with the plasmid-bornegene copy if the bacterial strain utilized is recombination-proficient.

[0050] Balanced-lethal systems based on catalytic enzyme production aresubject to a number of important deficiencies. In particular, sincecomplementation of the chromosomal gene deletion requires only a singlegene copy, it is inherently difficult to maintain more than a few copiesof an expression plasmid. The plasmidless host strain must be grown onspecial media to chemically complement the existing metabolicdeficiency.

[0051] Moreover, plasmidless cells may also benefit from “cross-feeding”effects when a diffusible growth factor is growth limiting.

[0052] There is therefore a need in the art for a Plasmid MaintenanceSystem which is not solely reliant on a balanced lethal system,particularly for use in bacterial live vector vaccines.

2. SUMMARY OF THE INVENTION

[0053] The present invention relates generally to a stabilizedexpression plasmid comprising a Plasmid Maintenance System and anucleotide sequence encoding a protein or peptide, such as a foreignantigen, and methods for making and using such stabilized expressionplasmids. The Plasmid Maintenance System of the present optimizesviability by using stabilized lower copy number expression plasmidscapable of expressing high levels of heterologous antigen in response toan environmental signal likely to be encountered in vivo after thevaccine organisms have reached an appropriate ecological niche.

[0054] In a particular aspect, the stabilized expression plasmid isemployed in a Salmonella typhi live vector vaccine, such as the strainCVD908-htrA.

[0055] The invention optimizes the maintenance of expression plasmids attwo independent levels by: (1) removing sole dependence on balancedlethal maintenance systems; and (2) incorporating a plasmid partitionsystem to prevent random segregation of expression plasmids, therebyenhancing their inheritance and stability. In one aspect of theinvention, the stabilized expression plasmid is recombinantly engineeredto express one or more antigens, preferably one or more Shiga toxin 2(Stx2) antigens or substantial homologues thereof, such as Shiga toxinsubunit pentamers or a genetically detoxified Stx 2.

[0056] The stabilized expression plasmid preferably comprises one ormore non-catalytic plasmid maintenance functions.

[0057] In another aspect, the expression plasmid comprises a PlasmidMaintenance System which comprises at least one PSK function and atleast one SEG function. For example, the Plasmid Maintenance System maycomprise a two-component Plasmid Maintenance System comprising one PSKfunction and one SEG function. Alternatively, the Plasmid MaintenanceSystem may comprise a three-component Plasmid Maintenance Systemcomprising a PSK function, a SEG function and another PSK. In apreferred alternative, the Plasmid Maintenance System compriseshok-sok+par+parA+phd-doc; wherein any of the stated functions may bereplaced by a substantial homologue thereof.

[0058] The Plasmid Maintenance Systems can be incorporated intomulticopy expression plasmids encoding one or more proteins or peptidesof interest. Such multicopy expression plasmids produce a gene dosageeffect which enhances the level of expression of the protein or peptideof interest. Where the Plasmid Maintenance System is to be employed in abacterial live vector vaccine, the protein or peptide of interest is oneor more foreign antigens.

[0059] In one aspect, the expression plasmid is a vaccine expressionplasmid comprising a Plasmid Maintenance System and at least oneantigen, for example, at least one Shiga toxin 2 (Stx2) antigen and/orsubstantial homologue thereof. Where the antigen is a Shiga toxin 2antigen, the Shiga toxin 2 antigen can, for example, be either a Bsubunit pentamer or a genetically detoxified Stx 2.

[0060] In another aspect the expression plasmid comprises a PlasmidMaintenance System which incorporates the ssb balanced lethal system andthe ssb locus of the bacterial live vector has been inactivated using asuicide vector comprising a temperature sensitive origin of replication.In one aspect, the bacterial live vector is S. typhi and the suicidevector is used to inactivate the ssb locus of S. typhi. In one aspect,the suicide vector is a derivative of pSC101 which carries sacB,described herein.

[0061] In another aspect, the present invention provides a PlasmidMaintenance System incorporating a PSK function involving a silentplasmid addiction system based on antisense RNA control mechanisms thatonly synthesize lethal proteins after plasmid loss has occurred.

[0062] In one aspect the expression plasmid comprises a series ofexpression plasmids, each comprising self-contained genetic cassettesencoding regulated expression of a heterologous antigen, an origin ofreplication, and a selectable marker for recovering the plasmid.

[0063] In one aspect the expression plasmid comprises a PlasmidMaintenance System which incorporates a PSK function based on the ssbgene. In a related aspect, mutated alleles such as ssb-1, describedherein, are incorporated into the expression plasmids to enhance highercopy number plasmids by over-expression of SSB1-like proteins to formthe required biologically active tetramers of SSB.

[0064] In another aspect, the expression plasmid comprises a promoter.The promoter is preferably an inducible promoter, such as the ompCpromoter. In one aspect, the inducible promoter is the mutatedP_(ompC1), or the P_(ompC3) promoter described herein.

[0065] In one aspect, the expression plasmid of the present inventioncomprises a plasmid inheritance (or partition) locus; an origin ofreplication selected to provide copy number which effectively stabilizesa given antigen; a PSK function; and a nucleotide sequence encoding anantigen and a promoter which ultimately controls translation of theantigen and has a strength which is selected to improve antigenproduction without killing the cell.

[0066] The present invention also provides a method of using theexpression plasmid comprising transforming a bacterial cell using saidexpression plasmid, and culturing the bacterial cell to produce theprotein or peptide (e.g., the antigen), and/or administering saidtransformed cell or cell culture to a subject. Where the transformedbacterial cells are administered to a subject, they are administered inan amount necessary to elicit an immune response which confers immunityto the subject for the protein or peptide. The subject is preferably ahuman, but may also be another animal, such as a dog, horse, or chicken.

[0067] In one aspect, an expression plasmid is provided which comprisesat least 3 independently functioning expression cassettes wherein onecassette encodes a protein or peptide of interest and the remainingcassettes each encode a different Plasmid Maintenance Function.

[0068] In one aspect, an expression plasmid is provided which encodes(1) a test antigen operably linked to a promoter and (2) a PlasmidMaintenance System.

[0069] In another aspect, a regulated test antigen expression cassetteis provided which operates such that as induction of antigen expressionis increased, a metabolic burden is placed on the bacterium which leadsphenotypically to plasmid instability, i.e. a selective advantage iscreated for all bacteria which can spontaneously lose the offendingplasmid. The test antigen can be the green fluorescent protein (GFPuv).The expression cassette encoding the test antigen can also comprise aninducible promoter, such as the ompC promoter, positioned such that theinducible promoter ultimately drives the translation of the testantigen.

[0070] In one aspect, a method of making an expression plasmid isprovided which comprises synthesizing an expression plasmid comprisingat least 3 independently functioning expression cassettes wherein onecassette encodes a protein or peptide of interest and the remainingcassettes each encode a different Plasmid Maintenance Function.

[0071] In one aspect, a method of screening Plasmid Maintenance Systemsis provided comprising: providing one expression cassette which encodesa protein or peptide of interest, and at least two other expressioncassettes, each encoding and capable of expressing in the host bacteriallive vector a different Plasmid Maintenance Function; inserting thethree expression cassettes into a single expression plasmid;transforming a bacterial live vector with the single expression plasmid;culturing the transformed bacterial live vector; and determining therate of introduction of plasmidless cells into the culture.

[0072] In one aspect, the present invention comprises an attenuatedbacterial live vector vaccine comprising an attenuated bacterial livevector which has been transformed with a stabilized expression plasmidcomprising a Plasmid Maintenance System, preferably a non-catalyticplasmid maintenance system.

[0073] In one aspect, the present invention comprises an attenuatedbacterial live vector vaccine comprising an attenuated bacterial livevector which has been transformed with an expression plasmid comprisinga Plasmid Maintenance System which incorporates at least one PSK systemand at least one SEG system. The attenuated bacterial live vector can,for example, be S. typhi CVD908-htrA.

[0074] The present invention also provides a method for vaccinating asubject comprising administering to the subject an amount of a bacteriallive vector vaccine sufficient to elicit an enhanced immune response.The present invention also provides a method for preventing a disease byvaccinating a subject using an amount of such bacterial live vectorsufficient to elicit a protective immune response to one or morepathogens of such disease. The subject is preferably a human but mayalso be another animal, such as a horse, cow or pig. For example, thepresent invention provides a method for preventing hemolytic uremicsyndrome (HUS) caused by Shiga toxin 2-producing enterohemorrhagicEscherichia coli by administering to a subject an amount of a bacteriallive vector transformed with a stabilized plasmid encoding at least oneShiga toxin 2 antigen.

[0075] In another aspect, the present invention provides a method forscreening Plasmid Maintenance Systems for efficacy, the methodcomprising: providing expression plasmids comprising the PlasmidMaintenance Systems described herein and encoding for a protein orpeptide of interest, said expression plasmids having copy numbers whichvary from low copy number (e.g. ˜5 copies per cell) to medium copynumber (e.g. ˜15 copies per cell) to high copy number (e.g. ˜60 copiesper cell); transforming bacterial live vectors with such expressionplasmids; and testing for rate of introduction of plasmidless cellsand/or rate of growth of plasmid-containing cells. The modified originsof replication may be origins of replication from the plasmids pSC101(low copy number), pACYC184 (medium copy number), and pAT153 (high copynumber). Independently functioning plasmid replication cassettes can beutilized which permit testing of the efficiency of one or more plasmidstabilization systems as copy number is increased.

[0076] In another aspect, the present invention provides stabilizedexpression plasmids for use in attenuated S. typhi live vectors whichcontain a selectable marker which can readily be replaced by a non-drugresistant locus or by a gene encoding an acceptable drug resistancemarker such as aph encoding resistance to the aminoglycosides kanamycinand neomycin.

[0077] The Plasmid Maintenance Systems of the present invention provideimproved stability of recombinant plasmids, overcoming prior artproblems of plasmid instability, for example, in bioreactor and livevector vaccination uses. The plasmids of the present invention arespecifically tailored for vaccine applications though such plasmids arealso useful in large scale protein production.

[0078] The plasmids of the present invention are a major improvementover the prior art in that they overcome the problems associated withplasmidless takeover and plasmid instability and have wide rangingutility in fields such as commercial protein production and attenuatedbacterial live vector vaccine production.

[0079] There has long been a need for a solution to the problems ofplasmidless takeover and plasmid stability associated with the field ofvaccine delivery and protein production. The present invention solvesthis long felt need.

3. DEFINITIONS

[0080] The term “Plasmid Maintenance System” (“PMS”) as used hereinrefers to a nucleotide sequence comprising at least onepost-segregational killing function (“PSK”) and at least onepartitioning or segregating system (“SEG”), and optionally including anyother Plasmid Maintenance Function.

[0081] The term “Plasmid Maintenance Function” is used herein to referto any plasmid-stability enhancing function associated with a PMS. Theterm includes both naturally-occuring nucleotide sequences encodingplasmid maintenance functions, as well as nucleotide sequences which aresubstantially homologous to such naturally-occurring plasmid maintenancefunctions and which retain the function exhibited by the correspondingnaturally-occurring plasmid maintenance function.

[0082] The term “Post-Segregational Killing System” (PSK) is used hereinto refer to any function which results in the death of any newly dividedbacterial cell which does not inherit the plasmid of interest, andspecifically includes balanced-lethal systems such as asd or ssb,proteic systems such as phd-doc, and antisense systems such as hok-sok.The term includes both naturally-occuring nucleotide sequences encodingsuch PSKs, as well as nucleotide sequences which are substantiallyhomologous to such naturally-occurring nucleotide sequences and whichretain the function exhibited by the corresponding naturally-occurringnucleotide sequences.

[0083] The term “substantially homologous” or “substantial homologue,”in reference to a nucleotide sequence or amino acid sequence, indicatesthat the nucleic acid sequence has sufficient homology as compared to areference sequence (e.g., a native sequence) to permit the sequence toperform the same basic function as the corresponding reference sequence;a substantially homologous sequence is typically at least about 70percent sequentially identical as compared to the reference sequence,typically at least about 85 percent sequentially identical, preferablyat least about 95 percent sequentially identical, and most preferablyabout 96, 97, 98 or 99 percent sequentially identical, as compared tothe reference sequence. It will be appreciated that throughout thespecification, where reference is made to specific nucleotide sequencesand/or amino acid sequences, that such nucleotide sequences and/or aminoacid sequences may be replaced by substantially homologous sequences.

[0084] The terms “Segregating System” and/or “Partitioning System” (bothreferred to herein as “SEG”) are used interchangeably herein to refer toany plasmid stability-enhancing function that operates to increase thefrequency of successful delivery of a plasmid to each newly dividedbacterial cell, as compared to the frequency of delivery of acorresponding plasmid without such a SEG system. SEG systems include,for example, equipartitioning systems, pair-site partitioning systems,and the par locus of pSC101. The term includes both naturally-occuringnucleotide sequences encoding such SEG systems, as well as nucleotidesequences which are substantially homologous to such naturally-occurringnucleotide sequences and which retain the function exhibited by thecorresponding naturally-occurring nucleotide sequences.

[0085] The term “detoxified” is used herein to describe a toxin havingone or more point mutations which significantly reduce the toxicity ofthe toxin as compared to a corresponding toxin without such pointmutations.

[0086] The term “immunizingly effective” is used herein to refer to animmune response which confers immunological cellular memory upon thesubject, with the effect that a secondary response (to the same or asimilar toxin) is characterized by one or more of the followingcharacteristics: shorter lag phase in comparison to the lag phaseresulting from a corresponding exposure in the absence of immunization;production of antibody which continues for a longer period thanproduction of antibody for a corresponding exposure in the absence ofsuch immunization; a change in the type and quality of antibody producedin comparison to the type and quality of antibody produced from such anexposure in the absence of immunization; a shift in class response, withIgG antibodies appearing in higher concentrations and with greaterpersistence than IgM; an increased average affinity (binding constant)of the antibodies for the antigen in comparison with the averageaffinity of antibodies for the antigen from such an exposure in theabsence of immunization; and/or other characteristics known in the artto characterize a secondary immune response.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIGS. 1A-1C: Genetic maps of exemplary pGEN expression plasmids(pGEN2, pGEN3, and pGEN4) of the present invention.

[0088] FIGS. 2A-2D: Genetic maps of exemplary oriE1-based expressionplasmids (pJN72, pJN51, pJN10, and pJN12) of the present invention.

[0089]FIG. 3: Flow cytometry histograms of GFP flourescence for CVD908-htrA carrying expression vectors with the hok-sok post-segregationalkilling system.

[0090] FIGS. 4A-4B: pGEN2 nucleotide sequence 1-4199.

[0091]FIG. 5: pGEN3 nucleotide sequence 1201-2400 showing the sequenceof ori15A.

[0092]FIG. 6: pGEN4 nucleotide sequence 1201-3850 showing the sequenceof ori101.

[0093] FIGS. 7A-7E: Genetic maps of exemplary ori15A-based pGENexpression plasmids (pGEN91, pGEN111, pGEN121, pGEN193, and pGEN222) ofthe present invention.

[0094]FIG. 8: Flow cytometry histograms of GFP flourescence forexpression plasmids pGEN91, pGEN111, pGEN121, pGEN193, and pGEN222.

5. DETAILED DESCRIPTION OF THE INVENTION

[0095] Bacterial live vector vaccines employ a bacterial live vector toexpress genes encoding protective antigens of bacterial, viral orparasitic pathogens. The bacterial protective antigens are preferablynon-native to the bacterial live vector, i.e. heterologous. Thebacterial live vector vaccine is administered to a host, therebyexposing the expressed antigens to the host's immune system, elicitingan immune response of appropriate character to confer immunity to thehost.

[0096] In order to achieve enhanced immunogenicity, the plasmidsexpressing such protective antigens must be stabilized. To theinventor's knowledge, no currently available S. typhi-based PlasmidMaintenance System takes advantage of naturally occurring partitionmechanisms known to improve the stability of multicopy plasmids in otherstrains.

[0097] The present invention provides a non-catalytic PlasmidMaintenance System for the stabilization of expression plasmids encodingforeign antigens in a S. typhi live vector vaccine strain. In one aspectthe S. typhi strain is CVD 908-htrA. In another aspect, the presentinvention improves and/or optimizes maintenance of expression plasmidsby providing Plasmid Maintenance Systems which operate at twoindependent levels: (1) removing sole dependence on catalytic balancedlethal maintenance systems; and (2) incorporating a plasmid partitionsystem which will prevent random segregation of the expression plasmids,thereby enhancing their inheritance and stability. A critical reason forpursuing this particular approach is that this method of improvingplasmid maintenance involves no additional manipulations of the livevector strain, and therefore can improve the immunogenicity ofheterologous antigens expressed within any live vector strain.

[0098] The non-catalytic Plasmid Maintenance System of the presentinvention improves the stability of multicopy expression plasmids withina bacterial live vector vaccine, such as CVD908-htrA.

[0099] In one aspect, the present invention incorporates the naturallyoccurring PSK function hok-sok from the antibiotic-resistance factorpR1, or a substantial homologue thereof, within multicopy expressionplasmids. The hok-sok system is a silent plasmid addiction system basedon antisense RNA control mechanisms that only results in synthesis oflethal proteins after plasmid loss has occurred.

[0100] The present invention also provides a plasmid maintenance systemcomprising a complementation-based PSK function in which the chromosomalgene ssb, encoding the essential non-catalytic single-stranded bindingprotein (SSB) required for DNA replication, is specifically deleted andinserted within a multicopy expression plasmid.

[0101] The present invention also provides an improved PlasmidMaintenance System comprising an expression plasmid encoding at leastone SEG locus and at least one PSK function.

[0102] 5.1 Suicide Vectors

[0103] Heterologous antigens can be expressed within live vectorstrains, such as CVD908-htrA, from genes residing either on plasmids orintegrated within the chromosome. One technique for integrating thesegenes into the host chromosome involves the use of temperature sensitive“suicide vectors” such as pIB307 which contains a temperature-sensitiveorigin of replication from pSC101 (ori101^(ts)). The present inventionprovides an improved suicide vector for use in CVD908 and CVD908-htrA,derived from pIB307 which allows for easier construction of mutagenesiscassettes to alter the live vector chromosome.

[0104] Integration of these suicide vectors into the chromosome byhomologous recombination results from temperature inactivation of theplasmid replication protein, RepA, a protein essential to the functionof ori101. Spontaneous resolution of the resulting unstable merodiploidintermediates is detected by counter-selection for loss of the sacB genecontained on the resolving suicide vector. The sacB gene contained onall excised plasmids encodes the levansucrase enzyme, which is lethalwhen expressed within the cytoplasm of enteric bacteria, including S.typhi, growing in the presence of sucrose. Since resolving merodiploidsare selected by incubating in the presence of 10% sucrose, excisedplasmids will kill host bacteria unless they cure spontaneously.

[0105] This system was successfully used to integrate akanamycin-resistance cassette into the ΔaroC1019 locus of CVD908.However, these experiments were successful because the gene beingmobilized into the chromosome of S. typhi encoded a selectabledrug-resistance marker. Using these early vectors, replacing thekanamycin-resistance cassette with a non-selectable marker was notsuccessful because, although the incoming marker could be integratedinto the chromosome as a merodiploid, resolution of the merodiploid toreplace the drug resistance gene was never detected.

[0106] The present invention also provides a method for using suchsuicide vectors to inactivate the ssb locus of attenuated Salmonellatyphi strains such as CVD908-htrA.

[0107] The present invention allows such suicide vectors to permitefficient mobilization of genes expressing proteins or peptides ofinterest, such as heterologous antigens, into the chromosome of S. typhiCVD908-htrA in two stages. For example, the present inventor introduceda sacB-aph cassette into the ΔaroC1019 locus, which was then selectedusing kanamycin. Generation of this S. typhiCVD908-htrAΔaroC1019::sacB-aph strain produced a valuable intermediatestrain into which, in theory, any structural gene can be efficientlyinserted into the aroC locus by marker-exchange. The sacB gene is usedas a counter-selectable marker by passing merodiploids in the presenceof 10% sucrose to select for replacement of the sacB-aph cassette withthe incoming antigen cassette, since resolution of merodiploids in thepresence of sucrose will result in loss of the sacB gene, in order toproduce viable progeny. This intermediate strain was employed toefficiently integrate the non-toxigenic mutant LT-K63 of the E. coliheat-labile enterotoxin, creating CVD908ΔaroC1019::LT-K63.

[0108] 5.2 Plasmid-Based Expression of Heterologous Antigens

[0109] Although chromosomal integration of foreign genes confersstability to such sequences, the genetic manipulations involved can bedifficult, and the drop in copy number of the heterologous gene oftenresults in production of insufficient levels of heterologous antigen toensure an optimal immune response.

[0110] In contrast, plasmid stability is a complex phenomenon whichdepends on multiple factors including (1) copy number of the plasmid;(2) appropriately regulated expression of genes contained within theplasmid; and (3) selective pressure for ensuring the proper segregationand inheritance of the plasmid.

[0111] To ensure stability, plasmids must be replicated in a regulatedmanner to prevent their copy number from rising to lethal levels.

[0112] In addition, plasmids must segregate during the division of agrowing bacterium to ensure that each daughter cell receives at leastone copy of the plasmid. Segregation can be a passive, random event oran active process involving synthesis of novel proteins which aid inplasmid segregation and inheritance. Successful inheritance of randomlysegregating plasmids relies on a high enough copy number of randomlydistributed plasmids within a dividing bacterium to virtually guaranteeinheritance of at least one plasmid by each daughter cell.

[0113] The commonly used plasmid cloning vectors, including medium copynumber pBR322 derivatives and high copy number pUC plasmids, areinherited by random segregation.

[0114] Active segregation involves the synthesis of proteins which areproposed to bind to such plasmids and further coordinate with themembranes of dividing bacteria to ensure that each daughter receives atleast one plasmid copy. Plasmids employing such active partitioningsystems are typically very low copy number plasmids such as the F sexfactor of E. coli or antibiotic resistance R-factors such as pR1 andpRK2.

[0115] The present invention exploits naturally occurring SEG functionsto enhance inheritance of multicopy expression plasmids, which wouldotherwise be inherited by random segregation, to increase the stabilityof these plasmids.

[0116] The present invention also takes advantage of other naturallyoccurring genetic systems in which daughter cells which do notsuccessfully inherit an expression plasmid will be killed and removedfrom the growing population, i.e., PSK functions. The incorporation ofmore than one category of plasmid stabilization function is referred toherein as a Plasmid Maintenance System. For example, the incorporationof both a SEG function such as a partition locus and a PSK function intoa single expression plasmid yields a Plasmid Maintenance System.

[0117] It should be noted that a gene conferring resistance to abactericidal antibiotic, such as the aph gene encoding resistance tokanamycin and neomycin, is also considered a PSK function, as is theasd-based balanced-lethal system.

[0118] 5.3 Balanced Lethal Systems

[0119] One method of ensuring the inheritance of expression plasmidsinvolves the construction of a PSK system or a substantial homologuethereof, referred to as a balanced lethal system, for plasmidsexpressing heterologous antigens. In a plasmid-based balanced lethalsystem, plasmids replicating in the cytoplasm of the bacterium express acritical protein required by the bacterium to grow and replicate. Lossof such plasmids removes the ability of the bacterium to express thecritical protein and results in cell death.

[0120] The asd system has recently been introduced into attenuated S.typhi vaccine strains in an attempt to increase the stability ofplasmids expressing synthetic hepatitis B viral peptides.

[0121] However, when volunteers were immunized with these live vectorstrains, no immune response to the foreign antigen was detected. SeeTacket et al., Infection and Immunity, 65:3381, 1997 (incorporatedherein by reference). In fact, to date, few reports have documented animmune response to plasmid-based expression of a foreign antigen fromplasmids (stabilized or otherwise) after vaccination of humans with anattenuated S. typhi live vector.

[0122] Although in some cases failure of live vector strains may haveresulted from over-attenuation of the strain itself, the inventor'sconclusion is that currently used PSK functions for plasmids suffer fromadditional limitations, in particular, from segregation limitations andcatalytic activity limitations. The present invention provides improvedexpression plasmids comprising enhanced segregation capabilities byincorporating at least one partitioning system along with at least onePSK system.

[0123] 5.4 Segregation Limitations

[0124] One limitation of plasmid maintenance functions such as the asdfunction (as well as the thyA function) is that they do not enhance theinheritance of resident plasmids, which continue to segregate randomlywith or without the presence of the asd function. Therefore, if residentexpression plasmids carrying asd genes are inherently unstable, theywill be lost, regardless of the requirement of the bacterium for Asd.

[0125] The inherent stability of an asd expression plasmid can bedefined by growing plasmid-bearing strains in the presence of DAP, whichremoves the selective pressure that ensures that all viable bacteriacontain the expression plasmid. If a given plasmid is inherentlyunstable, it will be lost from bacteria at a high rate and suchplasmidless bacteria will lyse in the absence of growth supplements; theoverall result of this effect will be a population of bacteria thatgrows much slower than wildtype unaltered strains.

[0126] The present invention improves plasmid stability by incorporatinga SEG function, such as a partition locus, or a substantial homologue ofa SEG function, onto the expression plasmid to enhance the inheritanceof such plasmids by actively dividing bacteria. Partition loci arenaturally present on the virulence plasmids of S. typhimurium. Tinge andCurtiss, Journal of Bacteriology, 172:5266, 1990 (incorporated herein byreference) reported that such partition loci were well conserved amongS. typhimurium virulence plasmids, and that when a 3.9 kb restrictionfragment encoding this locus was introduced onto the lower copy numberplasmid pACYC184 (˜15 copies per cell), the observed plasmid stabilityincreased from 34% plasmid-containing cells to 99% plasmid-bearing cellsafter 50 generations. The nucleotide sequence of this locus was laterdetermined by Cerin and Hackett, Plasmid, 30:30, 1993 (incorporatedherein by reference), (GenBank Accession Number M97752).

[0127] 5.5 Catalytic Activity Limitations

[0128] Another potential limitation of a plasmid maintenance functionsuch as the asd function (as well as the thyA system) is its reliance onan enzyme with catalytic activity. Given that complementation with onlya single copy of the asd gene is sufficient to remove auxotrophy, it isnot clear why all copies of a multicopy plasmid should remain stable,especially if they encode an especially problematic heterologous antigenwhich inhibits growth of the bacterium.

[0129] Further, although higher copy number expression plasmids mayexpress appreciable levels of a given heterologous antigen in vitro,such plasmids may not be maintained at the expected copy numbers in vivodue to toxicity and may in fact be present at much lower copy numbers,which would be expected to reduce any observed immune response specificfor the heterologous antigen. Accordingly, the present invention thusprovides stably maintained low and medium copy number plasmids forexpressing heterologous antigens.

[0130] 5.6 The Non-Catalytic ssb PSK Function

[0131] The potential limitation of catalytic activity associated withbalanced lethal systems is addressed here through the use of plasmidsexpressing the single-stranded binding protein (SSB) from S. typhi totrans-complement an otherwise lethal mutation introduced into thechromosomal ssb gene. The biochemistry and metabolic roles of the E.coli SSB protein have been extensively reviewed in Lohman et al., AnnualReviews in Biochemistry 63:527, 1994 and Chase et al., Annual Reviews inBiochemistry 55:103, 1986 (the disclosures of which are incorporatedherein by reference).

[0132] SSB is a non-catalytic 177 amino acid protein, with a relativemolecular weight of 19 kDa, that binds with high affinity tosingle-stranded DNA (ssDNA), and plays an essential role as an accessoryprotein in DNA replication, recombination, and repair. The biologicallyrelevant form of SSB involved in binding to ssDNA is a tetramer, whichbinds in two modes to ssDNA, intimately associating with an average ofeither 35 (SSB₃₅-binding mode) or 65 bases (SSB₆₅-binding mode). Thespecific conditions controlling the preferred mode of binding arecomplex and depend on the surrounding concentration of monovalent anddivalent salts, pH, and temperature, as well as the amount of SSBprotein present. Under given conditions, high concentrations of SSBfavor the SSB₃₅-binding mode, with lower SSB concentrations favoring theSSB₆₅-mode. However, it must be emphasized that in both binding modes,the required conformation of SSB is a tetramer.

[0133] Spontaneously occurring temperature-sensitive point mutationswithin the ssb gene have now been characterized at the biochemical,physiological, and nucleotide level; one such mutant, ssb-1, containsthe point mutation His 55 to Tyr, and has been found to be unable toassemble correctly into tetramers at non-permissive temperatures andnatural expression levels. These mutant strains exhibittemperature-sensitive lethal defects in DNA replication andrecombination.

[0134] The segregation frequencies of plasmids carrying ssb whichcomplement chromosomal ssb mutations in E. coli bacteria were examinedby Porter et al. Bio/Technology 8:47, 1990 (incorporated herein byreference). They observed that in experiments involving bioreactors, thesegregation frequency in plasmid-bearing strains growing in continuousculture under non-selective conditions for 150 hours was less than1×10⁻⁷; this segregation frequency was independent of copy number, asboth lower copy number pACYC184 plasmids and very high copy number pUC19plasmids were maintained at the same frequency. However, it must benoted that the plasmids involved expressed only a drug-resistance markerin addition to the SSB protein.

[0135] The present invention provides an improved plasmid maintenancesystem which incorporates a partition locus such as that present onpSC101, or a substantial homologue of such partition locus, and may alsoincorporate an active partitioning system, or a substantial homologuethereof, such as that described above for the virulence plasmid of S.typhimurium.

[0136] The present invention removes dependence on catalytic enzymes toconfer plasmid stability. In one aspect, mutated alleles similar tossb-1 are introduced into the expression plasmids to enhance higher copynumber plasmids by overexpression of SSB1-like proteins to form therequired biologically active tetramers of SSB. In another aspect thepresent invention provides a PSK function involving a silent plasmidaddiction system based on antisense RNA control mechanisms that onlysynthesize lethal proteins after plasmid loss has occurred.

[0137] 5.7 Expression Plasmids and Self-Contained Genetic Cassettes Thepresent invention also comprises a series of expression plasmids whichare referred to herein as pGEN plasmids. pGEN plasmids compriseself-contained genetic cassettes encoding regulated expression of aheterologous antigen, an origin of replication, and a selectable markerfor recovering the plasmid. This vector series has been specificallydesigned to test whether any Plasmid Maintenance System can increase thestability of plasmids, for example within an attenuated S. typhi vaccinebackground.

[0138] The basic structure of these vectors is represented in FIG. 1,and the composite gene sequence for the vector pGEN 2 is represented inFIG. 4; FIGS. 5 & 6 show specific composite sequences for the origins ofreplication in pGEN3 and pGEN4 respectively.

[0139] It is critical to note that the pGEN plasmids are designed tocomprise 3 independently functioning genetic cassettes. These cassetteshave been constructed such that individual components can be optimizedby replacement as necessary. Accordingly, in addition to the variousPlasmid Maintenance Systems described herein, the cassettes can testother promising systems now in existence or which may become availablein the future. Further, the optimized plasmid(s) can be adapted toexpress relevant protective heterologous antigens within attenuatedvaccine strains for immunization of humans.

[0140] The pGEN plasmids provide a regulated test antigen expressioncassette which operates such that as induction of antigen expression isincreased, a metabolic burden is placed on the bacterium which leadsphenotypically to plasmid instability, i.e. a selective advantage iscreated for all bacteria which can spontaneously lose the offendingplasmid. Thus one aspect of the present invention provides aconditionally unstable plasmid which can be examined for stability asplasmid maintenance systems are incorporated.

[0141] In a preferred mode, the regulated test antigen expressioncassette contained within the pGEN plasmids comprises the inducible ompCpromoter, or a substantial homologue thereof, driving expression of adetectable protein, such as the codon-optimized green fluorescentprotein (GFPuv, available from Clontech), overexpression of which istoxic to E. Coli and S. typhi.

[0142] The present invention also comprises a series of plasmidreplicons having copy numbers which vary from low copy number (i.e., ˜1to ˜10, preferably ˜5 copies per cell) to medium copy number (i.e., ˜11to ˜25, preferably ˜15 copies per cell) to high copy number (i.e., ˜26to ˜100, preferably ˜60 copies per cell). To accomplish this, origins ofreplication from the well-characterized plasmids pSC101, pACYC184, andpAT153 have been modified using polymerase chain reaction (PCR)techniques to create independently functioning plasmid replicationcassettes. These replication cassettes permit testing of the efficiencyof a plasmid maintenance system as copy number is increased.

[0143] The present invention also comprises selectable expressionplasmids for use in attenuated S. typhi live vectors. These expressionplasmids contain a selectable marker which can ultimately be replacedeither by a non-drug resistant locus, such as ssb, or by a gene encodingan acceptable drug resistance marker such as aph encoding resistance tothe aminoglycosides kanamycin and neomycin.

[0144] To accomplish this, resistance cassettes encoding resistance tocarbenicillin and tetracycline have been constructed, with transcriptionbeing efficiently terminated by an rrnB T1T2 terminator. A detaileddescription of the individual components comprising the expression andreplication cassettes follows.

[0145] Specific components of the Plasmid Maintenance System can besystematically inserted into the basic expression replicons to assessany individual or synergistic influence of these functions on plasmidstability in the presence and absence of selection. For example, apost-segregational killing function (e.g., the hok-sok locus) can beinserted as an EcoRI-XbaI cassette, such that flanking transcriptionfrom surrounding loci, such as the antigen and selection cassettes, isdivergent and will not significantly disturb the wild type transcriptionlevels which control the lethality of this locus (FIG. 7B, pGEN111).

[0146] Similarly, the par passive partition locus can be inserted as aBamHI-BglII fragment between the origin of replication and selectioncassettes (FIG. 7C, pGEN 121). Interestingly, in the work leading to thepresent invention, it was observed that the orientation of the par locusenhances synthesis of GFPuv on solid medium when inserted in the naturalorientation found within ori101of pSC101; this orientation was adoptedfor all of the expression plasmids.

[0147] The active partitioning locus is preferably the parA locus,constructed as an XhoI-EcoRI cassette from the same pR1 resistanceplasmid from which hok-sok was adapted. To preserve naturaltranscription levels and regulation within this locus, the cassette ispreferably positioned within an area of the expression plasmids suchthat flanking transcription progresses away from parA (FIGS. 7D and 7E,pGEN193 and pGEN222).

[0148] 5.8 Components of the Antigen Expression and ReplicationCassettes

[0149] 5.8.1 Promoter

[0150] It will be appreciated by one of skill in the art that a widevariety of components known in the art may be included in the expressioncassettes of the present invention, including a wide variety oftranscription signals, such as promoters and other sequences thatregulate the binding of RNA polymerase to the promoter. The operation ofpromoters is well known in the art and is described in Doi, Regulationof Gene Expression, Modern Microbial Genetics pages 15-39 (1991) (theentire disclosure of which is incorporated herein by reference). Theensuing description uses the ompC promoter by way of example, and is notmeant to delimit the invention.

[0151] The promoter is preferably an environmentally regulatablepromotor controlled by a biologically relevant signal such asosmolarity. In a preferred mode, the promoter is the ompC promoter. TheompC gene encodes a porin protein which inserts as a trimer into theouter membrane of a bacterial cell. Expression and control of ompC iscomplex and has recently been reviewed in considerable detail in Prattet al., Molecular Microbiology 20:911, 1996 and Egger et al., Genes toCells 2:167,1997 (the disclosures of which are incorporated herein byreference).

[0152] Synthesis of the OmpC protein is ultimately controlled at thelevel of transcription by the osmolarity of the surrounding environmentsuch that increases in osmolarity are accompanied by increases in thetranscription of ompC. However, increases in osmolarity do not directlymediate increases in the transcription of ompC. Rather, the bacteriumsenses the surrounding osmolarity using a two-component signaltransduction system encoded by the ompB operon. This operon is composedof two genes transcribed in the order envZ-ompR. The envZ gene encodes a450 amino acid (a.a.) protein, containing two transmembrane regions,which inserts into the bacterial inner membrane (perhaps as a dimer)with an N-terminal 118 a.a. osmotic-sensing domain extending into theperiplasmic space and a C-terminal 270 a.a. catalytic domain extendinginto the cytoplasm. The C-terminal catalytic domain possesses bothkinase and phosphatase activities which are modulated by osmolarity suchthat as osmolarity increases, kinase activity predominates, and asosmolarity drops, phosphatase activity predominates.

[0153] EnvZ kinase activity phosphorylates aspartic acid residue 55 ofthe 239 a.a. cytoplasmic protein OmpR, creating OmpR-P. It is the OmpR-Pmodified protein which binds to the ompC promoter and activatestranscription by RNA polymerase; therefore, as osmolarity increases,increasing kinase activity of EnvZ produces higher levels of OmpR-P,which in turn lead to greater transcription of ompC. OmpR-P binds to aregion of the ompC promoter spanning bases −41 (relative to thetranscriptional start site of +1) to −102, with initial binding ofOmpR-P to bases −78 through −102 being followed by additional binding tobases extending to −41 as the concentration of OmpR-P increases withosmolarity. In addition, OmpR-P has been shown to bind to an AT-richupstream region extending back to base −405 which further enhances ompCtranscription.

[0154] In a preferred embodiment the ompC promoter fragment from E. colispans nucleotides +70 through −389. This promoter can directtranscription within attenuated S. typhi strains of an antibioticresistance gene, such as the kanamycin resistance gene in an osmoticallysensitive manner. For example, our experiments have demonstrated thatwhen the concentration of NaCl in liquid growth medium was increasedfrom 0 mM to 300 mM, resistance to kanamycin increased from 0 μg/mlto >800 μg/ml.

[0155] 5.8.2 Origin of Replication

[0156] Due to varying degrees of toxicity associated with differentheterologous antigens (i.e. higher toxicity for antigens derived fromparasitic organisms such Plasmodium falciparum vs. virtually no toxicityfor the fragment C of tetanus toxin), the present invention provideslive vector vaccines which preferably express such antigens from eitherlow or medium copy plasmids. It will be appreciated by one skilled inthe art that the selection of an origin of replication will depend onthe degree of toxicity, i.e., the copy number should go down as toxicityto the bacterial strain goes up. In a preferred mode, the PlasmidMaintenance System(s) used are capable of stabilizing replicons of lowor medium copy numbers.

[0157] It is preferable for the origin of replication to confer anaverage copy number which is between about 2 and about 75. In apreferred mode the origin of replication is selected to confer anaverage copy number which is between about 5 and about 50. Morepreferably the range is from about 5 to about 30. Optimally, the rangeis from about 15 to about 20.

[0158] In one aspect, the origin of replication is from pSC101,conferring a copy number of approximately 5 per genome equivalent.

[0159] The oriE1 locus specifies synthesis of a 555 base transcriptcalled RNA I and synthesis of a 110 base antisense RNA transcript calledRNA II. As RNA I is synthesized, the 5′-proximal region of thetranscript adopts a stem-loop structure composed of 3 domains which canhybridize to a complementary stem-loop structure formed by RNA II,resulting in a double stranded RNA-RNA structure forming which causesplasmid replication to abort.

[0160] As synthesis of RNA I continues, generating the full-length 555base transcript, a rearrangement of the secondary structure of thetranscript destroys the initial 3 domain stem-loop structure to form analternate stem-loop configuration which no longer hybridizes to RNA II.Formation of this alternate structure allows the transcript to hybridizeto one DNA strand of the plasmid itself, forming an RNA-DNA complexwhich is nicked by endogenous RNAse H to trigger synthesis of the firstDNA strand of the plasmid and plasmid replication.

[0161] Plasmid replication is therefore controlled by synthesis of RNAI, which undergoes a cascade of structural configurations leading toinitiation of replication. The necessary progression of the RNA Ifolding cascade (and resulting replication initiation) is interrupted bycompetition of the domains with RNA II. This mechanism is essentiallythe same in plasmids containing either oriE1 or ori15A.

[0162] The reason these two types of plasmids can coexist within thesame bacterium is due to sequence divergence within the region ofhybridization between RNA I and RNA II, such that the RNA II from ori15Awill not hybridize to RNA I from oriE1; this sequence divergence alsoaffects the stability of the RNA I: RNA II hybrid, accounting for thedifferences in copy number between plasmids carrying the oriE1 or ori15Aorigins of replication.

[0163] The structural organization of the engineered origins ofreplication cassettes for pSC101 (ori101; ˜5 copies per genomeequivalent), pACYC184 (ori15A derivative; ˜15 copies per genomeequivalent), and pAT153 (oriE1 derivative; ˜60 copies per genomeequivalent) are analogous in structure and function.

[0164] 5.8.3 Expressed Protein or Peptide

[0165] When the expression cassette is used to screen PlasmidMaintenance Systems, it preferably expresses a protein or peptide withno metabolic activity. A preferred protein is the green flourescentprotein (GFP) of the bioluminescent jellyfish Aequorea victoria, a 238amino acid protein which undergoes a post-translational modification inwhich 3 internal amino acids (⁶⁵Ser-Tyr-Gly⁶⁷) are involved in acyclization and oxidation reaction. The resulting fluorophore emitsblue-green light maximally at a wavelength of 509 nm upon irradiationwith long-wave ultraviolet light at a wavelength of 395 nm. In addition,fluorescence activity is remarkably constant over a wide range of pHfrom 5.5-12 and at temperatures up to 70° C.

[0166] Since GFP has no known catalytic activity, the level of observedfluorescence within individual bacteria expressing GFP can provide adirect indication of transcription levels of the gfp gene carried byeach bacterium. Expression of the GFP protein has now been quantitatedin a variety of both prokaryotic and eukaryotic cells and requires noadditional cofactors or enzymes from A. victoria. Fluorophore formationis apparently dependent either on ubiquitous enzymes and cofactors, oris an autocatalytic event.

[0167] Individual bacteria expressing GFP can be quantitated eitheralone or within macrophages, epithelial cell lines, and infected animaltissues using flow cytometry. GFP fluorescence is absolutely dependenton residues 2-232 of the undenatured protein. However, fusion ofunrelated biologically active protein domains to the N-terminus of GFPhas still resulted in fusion proteins with the expected heterologousbiological activity which continue to fluoresce as well.

[0168] It has been confirmed by sequence analysis (Clontech) that thegfp allele preferred here (i.e. gfpuv) expresses a GFP mutant (GFPuv)containing 3 amino acid substitutions (not involving the fluorophore)which increase fluorescence 18-fold over that of wildtype GFP.

[0169] In addition, 5 rarely used arginine codons have been optimizedfor efficient expression of GFP in E. coli. Since comparison ofexpression levels of various heterologous proteins in E. coli and CVD908has demonstrated equivalent or superior expression within CVD908, it wasexpected that gfpuv will function efficiently in CVD908-htrA.

[0170] A coding sequence is inserted in a correct relationship to apromoter where the promoter and the coding sequence are so related thatthe promoter drives expression of the coding sequence, so that theencoded peptide or protein is ultimately produced. It will be understoodthat the coding sequence must also be in correct relationship with anyother regulatory sequences which may be present.

[0171] 5.8.4 Heterologous Antigens

[0172] The expression plasmids of the present invention preferablyexpress an antigen for presentation to a host to elicit an immuneresponse resulting in immunization and protection from disease. WhileShiga toxins are presented herein as examples of antigens usefullyexpressed by the vaccine expression plasmids disclosed herein, theinvention is broad in scope and encompasses the expression of anyantigen which does not destroy the bacterial live vector and whichelicits an immune response when the bacterial live vector containingsaid expression plasmid(s) is administered to a host, i.e., a human orother animal.

[0173] The vaccine expression plasmids provided herein are used togenetically transform attenuated bacterial strains, preferably strainsused for human vaccination and most preferably used to transformattenuated S. typhi vaccine strains such as CVD908-htrA, and preferablyencode either the B subunit of Stx2 or a genetically detoxified Stx2holotoxin.

[0174] A subset of STEC most often referred to as enterohemorrhagic E.coli (EHEC) are capable of causing severe clinical syndromes includinghemorrhagic colitis, hemolytic uremic syndrome (HUS) and thromboticthrombocytopenic purpura (TTP) in a small proportion of infectedindividuals, in addition to causing non-bloody diarrhea in most others.

[0175] Hemorrhagic colitis is characterized by copious bloody diarrhea,usually without fever or with only low-grade fever and a relativepaucity of fecal leukocytes demonstrable in the diarrheal stools. Thesefeatures differentiate hemorrhagic colitis from dysentery caused byShigella which is typically scanty stools of blood and mucus, precededby high fever and with large numbers of fecal leukocytes visible bymicroscopy.

[0176] HUS, a potentially fatal disease that most often affects youngchildren but may afflict individuals of any age, is characterized by thetriad of microangiopathic hemolytic anemia, thrombocytopenia and uremia.Currently in North America, HUS is the most frequent cause of acuterenal failure in infants and young children. In a study by Siegler etal. of 288 patients treated for postdiarrheal HUS in Utah from1970-1994, severe disease (defined as anuria lasting longer than 7 days,oliguria lasting for longer than 14 days, or extrarenal structuraldamage such as stroke) occurred in 25% of cases and was associated withchildren less than two years of age; about one third of these severecases of HUS resulted in death (5%) or severe sequelae includingend-stage renal disease (5%) or chronic brain damage (3-5%), with lesssevere chronic problems involving hypertension, proteinuria, orazotemia.

[0177] TTP, which most often affects adults, is characterized byneurologic complications such as stroke, in addition tothrombocytopenia, hemolytic anemia and renal disease.

[0178] By far the most common EHEC serotype is O157:H7. Nevertheless,other EHEC serotypes also cause HUS and hemorrhagic colitis, includingO26:H11, O111:H8 and a number of others. EHEC strains associated withHUS always elaborate one or more Shiga toxins and carry a 60 MDavirulence plasmid. In addition, most also harbor a chromosomalpathogenicity island (so-called LEE) having a set of genes that encodethe ability to attach and efface. It is well accepted that Shiga toxinselaborated by EHEC play a key role in the pathogenesis of hemorrhagiccolitis and HUS.

[0179] As described in detail below, the Shiga toxin family is comprisedof two groups of toxins, Stx1 (which is essentially identical tocytotoxin/neurotoxin/enterotoxin produced by Shigella dysenteriae type1, the Shiga bacillus) and Stx2 (which is immunologically distinct fromStx1 and has several related variants). In the USA, the overwhelmingmajority of EHEC associated with cases of HUS express Stx2, either aloneor in conjunction with Stx1.

[0180] The most important reservoir of EHEC infection are bovines. Thesingle most important mode of transmission of EHEC to humans is via theconsumption of under-cooked contaminated beef, most often ground beef.Less commonly, a variety of other food vehicles and other modes oftransmission have been incriminated. Most notably, EHEC are one of thehandful of bacterial enteric pathogens, which, like Shigella, can betransmitted by direct contact or by contact with contaminated fomites.

[0181] There is great anticipation and optimism on the part of mostepidemiologists that irradiation of meat sold in the USA willdrastically curtail the transmission of EHEC to humans, since it willcurtail the single most important mode of transmission. Nevertheless,certain risk groups exposed to other modes of transmission of EHEC willnot benefit from this intervention. For example, the exposure ofabattoir workers to EHEC, an occupational hazard, occurs at a point inthe meat processing cycle prior to when irradiation would be utilized.For such special groups such as these for whom risk will remain evenafter irradiation of meat becomes commonplace, anti-EHEC vaccines can beuseful. The present invention provides vaccines against EHEC useful forthe prevention of infection (in the animal reservoirs or in humans) andfor preventing the severe complications of EHEC infection by stimulatingneutralizing Shiga antitoxin.

[0182] Studies with attenuated Vibrio cholerae O1 expressing Stx1 Bsubunit have demonstrated the feasibility of eliciting neutralizingShiga antitoxin by mucosal immunization with live vectors. However,since virtually all EHEC associated with HUS cases in the USA expressStx2, alone or in conjunction with Stx1, it is preferable that a vaccinefor preventing the severe complications of EHEC infection viaelicitation of toxin-neutralizing antibodies should stimulate anti-Stx2as well as Stx1. It is within the broad scope of the present inventionto provide a stabilized plasmid system for expressing Stx2 antigens,alone or in conjunction with Stx1, in an attenuated S. typhi livevector.

[0183] Other antigens which may be suitably delivered according to thecompositions and methods of the present invention include, for example,those for hepatitis B, Haemophilus influenzae type b, hepatitis A,acellular pertussis (_(ac)P), varicella, rotavirus, Streptococcuspneumoniae (pneumococcal), and Neisseria meningitidis (meningococcal).See Ellis et al., Advances in Pharm., 39: 393423, 1997 (incorporatedherein by reference).

[0184] In one aspect, the antigens encoded by the expression plasmids ofthe present invention are cancer vaccines.

[0185] In another aspect, the antigens encoded by these plasmids aredesigned to provoke an immune response to autoantigens, B cell receptorsand/or T cell receptors which are implicated in autoimmune orimmunological diseases. For example, where inappropriate immuneresponses are raised against body tissues or environmental antigens, thevaccines of the present invention may immunize against the autoantigens,B cell receptors and/or T cell receptors to modulate the responses andameliorate the diseases. For example, such techniques can be efficaciousin treating myasthenia gravis, lupus erythematosis, rheumatoidarthritis, multiple sclerosis, allergies and asthma.

[0186] 5.8.4.1 The Shiga Toxin Family

[0187] Conradi in 1903 first reported that S. dysenteriae 1 produced apowerful exotoxin. Because injection of this toxin led to hind limbparalysis of rabbits it was originally called a neurotoxin. Subsequentlythis toxin, Shiga toxin, was shown to be lethal for certain cells intissue culture (i.e., it was a cytotoxin). Vicari et al. and then Keuschet al. demonstrated that it also functioned as an enterotoxin.

[0188] Scientists now recognize the existence of a family of Shigacytotoxins which inhibit protein synthesis, leading to cell death forsusceptible cells. For many years after the revelation that such toxinswere produced by certain E. coli strains in addition to the originalShiga toxin produced by Shigella dysenteriae type 1, the nomenclaturefor this family of toxins was confusing. Since early reports describedthe activity of these toxins on Vero cells (a cell line derived fromAfrican green monkey kidney epithelial cells), many investigators calledthem verotoxins. Others referred to these toxins expressed in E.coli asShiga-like toxins.

[0189] The protein toxins are collectively referred to herein as Shigatoxins (Stx), and the genes encoding these toxins are designated as stxwith subscripts denoting the group and variant [i.e. stx₁ for the Shigatoxin produced by E. coli that is essentially identical to that ofShigella dysenteriae type 1 (stx), and stx₂, Stx_(2c), Stx_(2d),Stx_(2e) for the antigenically distinct group of related toxins].

[0190] The structure, biochemistry and antigenicity of Shiga toxins arewell described in Melton-Celsa et al., Eschericia Coli 0157:H7 and OtherShiga Toxin-producing E. coli Strains, 1998; Takeda, Bacterial Toxinsand Virulence Factors in Disease, 1995; Gyles, Canadian J. ofMicrobiology, 38:734, 1992; and O'Brien et al., Current Topics inMicrobiology and Immunology, 180:165, 1992 (the disclosures of which areincorporated herein by reference).

[0191] These Shiga cytotoxins are composed of a single catalytic Asubunit of approximately 32 kDa non-covalently associated with apentameric receptor binding domain of approximately 7.7 kDa B subunits.These subunits are encoded by a single operon of the order stxA-stxB;transcription of the stx and stx₁ operons are iron-regulated in both S.dysenteriae type 1 and E. coli, but no environmental control signalshave as yet been determined for any stx₂ operon. None of these toxins isencoded on a plasmid; rather they are phage-encoded (Stx1, Stx2, Stx2c,and Stx2d) or are chromosomally encoded (Stx, Stx2e).

[0192] As mentioned above, all members of the Shiga toxin family arecytolytic toxins which inhibit protein synthesis within susceptiblecells by blocking the binding of elongation factor 1-dependentaminoacyl-tRNA to ribosomes. For all toxins identified from humaninfections, penetration of susceptible cells by endocytosis followsbinding of the holotoxin to the necessary cell surface glycolipidreceptor globotriaosyl ceramide (Gb₃), traffiking of the toxin to theGolgi apparatus and endoplasmic reticulum, followed by release into thecytoplasm. Shiga toxins are RNA N-glycosidases which depurinate a singleadenine from the 28S RNA of the eukaryotic 60S ribosomal subunit, thusinactivating the 60S subunit and eventually leading to cell death.

[0193] There are six prototypic members of the Shiga toxin family: Stx,Stx1, Stx2, Stx2c, Stx2d, and Stx2e, which differ from one anotherimmunologically and in toxic activity. Significant detail has beenincluded here to provide background for understanding the significanceof point mutations discussed below, which are required for thegenetically detoxified holotoxins. The members of the Shiga toxin familydiffer from one another in 3 fundamental ways, as recently summarized byMelton-Celsa et al., Eschericia coli 0157:H7 and Other Shigatoxin-producing E. coli strains, 1998.

[0194] (1) Immunologically: The Shiga toxin family is composed of twoserogroups, Stx/Stx1 and Stx2; antisera raised against Stx/Stx1 do notneutralize members of the Stx2 serogroup, as judged by the Vero cellcytotoxicity assay.

[0195] (2) Structurally: Stx and Stx1 are essentially identical,differing in a single amino acid at position 45 of the mature A subunit,and the crystal structure for the Stx holotoxin has been solved. Theprototype Stx2 is only 55% homologous to residues of the mature Asubunit of Stx/Stx1 and 57% homologous to the mature B subunit, whichexplains why antisera raised against Stx/Stx1 do not neutralize membersof the Stx2 group. Within the Stx2 group, Stx2e is most distantlyrelated, sharing 93% amino acid homology to the mature A subunit of Stx2and 84% homology to the mature B subunit; Stx2c and Stx2d are verysimilar to Stx2, sharing 99-100% homology in mature A subunit residuesand 97% homology in mature B subunit residues.

[0196] (3) Cytotoxicity: Stx2 is among the most lethal of the Shigatoxins, with an LD₅₀ for mice injected intraperitoneally of 0.5-2 ng.The LD₅₀ for Stx1 and Stx2e is 200-400 ng, and 1-5 ng for Stx2d;however, Stx2d is unusual in that this toxin can become activated bymurine intestinal mucus to increase the toxicity of the toxin, loweringthe LD₅₀ to 0.5 ng.

[0197] 5.8.5 Site-Specific Mutagensis of Shiga Toxins

[0198] In one aspect, the invention provides a genetically detoxifiedShiga toxin. The detoxification is accomplished by site-specificmutagenesis, introducing two defined and well-separated point mutationsaltering critical residues within the catalytic site of the A subunit.The invention also introduces two additional defined and well-separatedpoint mutations within the B subunit to alter critical residues withinthe primary binding site (i.e. SITE I) residing within the cleft formedby adjacent B subunits of the holotoxin pentameric ring.

[0199] Prior attempts have been made to alter the lower affinity bindingSITE II. However, this binding site has only been identified frommolecular modeling studies, and is not extensively supported bymutational studies which favor SITE I binding of the Gb₃ receptor. Evenif SITE II is an alternate low-affinity binding site allowing entry ofour mutant holotoxin into susceptible cells, the inactivation of thecatalytic domain will still prevent cell death.

[0200] Based on amino acid sequence alignments, X-ray crystallographystudies, and molecular modeling studies, essential amino acids have beenidentified comprising the active site within the catalytic A subunit ofStx, as well as those residues comprising the binding SITE I within theB subunit pentamer of Stx/Stx1. It is the inventor's conclusion that theamino acids essential to the active site are selected from the groupconsisting of Tyr 77, Tyr 114, Glu 167, Arg 170, and Trp 203. Theresidues believed to be required for receptor binding to the cleftsformed by adjacent B subunits include Lys 13, Asp 16, Asp 17, Asp 18,Thr 21, Glu 28, Phe 30, Gly 60, and Glu 65. These site predictions areconsistent with functional studies and in vivo experiments using definedsingle and double mutations, within individual domains of the holotoxin,introduced by site-specific mutagenesis. A summary of such mutations ispresented in Table 1. Based on these data and crystallographicpredictions, it is within the broad practice of the invention to provideexpression plasmids encoding Shiga toxins having two specific sets ofpoint mutations within both the A and B subunits to create non-toxicmutant Stx2 holotoxins for use as vaccines, such as by expression withinattenuated S. typhi live vectors such as CVD908-htrA. TABLE 1 SITESPECIFIC MUTAGENESIS STUDIES DROP IN DROP IN NEUTRALIZING SUBUNIT TOXINMUTATION CYTOTOXICITY LETHALITY ANTIBODIES A Stx1 Leu201 → Val + NOcytotoxicity — — of residues 202-213 Stx1 Glu167 → Asp 10³ — — Stx1Arg170 → Leu 10³ — — Stx2 Glu167 → Asp 10³ — — Stx2e Glu167 → Asp 10⁴ —— Stx2e Arg170 → Lys 10  — — Stx2e Glu167 → Asp 10⁴ — — Arg170 → LysStx2e Glu167 → Gln 10⁶  10⁴ Y B Stx Asp16 → His + NO cytotoxicity — —Asp17 → His Stx Arg33 → Cys 10⁸ — — Stx Gly60 → Asp 10⁶ — — Stx1 Phe30 →Ala 10⁵ 10 Y Stx2 Ala42 → Thr 10³-10⁴ Y Y Stx2 Gly59 → Asp 10³-10⁴ Y Y

[0201] 5.9 Pharmaceutical Formulations

[0202] It is contemplated that the bacterial live vector vaccines of thepresent invention will be administered in pharmaceutical formulationsfor use in vaccination of individuals, preferably humans. Suchpharmaceutical formulations may include pharmaceutically effectivecarriers, and optionally, may include other therapeutic ingredients,such as various adjuvants known in the art.

[0203] The carrier or carriers must be pharmaceutically acceptable inthe sense that they are compatible with the therapeutic ingredients andare not unduly deleterious to the recipient thereof. The therapeuticingredient or ingredients are provided in an amount and frequencynecessary to achieve the desired immunological effect.

[0204] The mode of administration and dosage forms will affect thetherapeutic amounts of the compounds which are desirable and efficaciousfor the vaccination application. The bacterial live vector materials aredelivered in an amount capable of eliciting an immune reaction in whichit is effective to increase the patient's immune response to theexpressed mutant holotoxin or to other desired heterologous antigen(s).An immunizationally effective amount is an amount which confers anincreased ability to prevent, delay or reduce the severity of the onsetof a disease, as compared to such abilities in the absence of suchimmunization. It will be readily apparent to one of skill in the artthat this amount will vary based on factors such as the weight andhealth of the recipient, the type of protein or peptide being expressed,the type of infecting organism being combatted, and the mode ofadministration of the compositions.

[0205] The modes of administration may comprise the use of any suitablemeans and/or methods for delivering the bacterial live vector vaccinesto a corporeal locus of the host animal where the bacterial live vectorvaccines are immunostimulatively effective.

[0206] Delivery modes may include, without limitation, parenteraladministration methods, such as subcutaneous (SC) injection, intravenous(IV) injection, transdermal, intramuscular (IM), intradermal (ID), aswell as non-parenteral, e.g., oral, nasal, intravaginal, pulmonary,opthalmic and/or rectal administration.

[0207] The dose rate and suitable dosage forms for the bacterial livevector vaccine compositions of the present invention may be readilydetermined by those of ordinary skill in the art without undueexperimentation, by use of conventional antibody titer determinationtechniques and conventional bioefficacy/biocompatibility protocols.Among other things, the dose rate and suitable dosage forms depend onthe particular antigen employed, the desired therapeutic effect, and thedesired time span of bioactivity.

[0208] The bacterial live vector vaccines of the present invention maybe usefully administered to the host animal with any other suitablepharmacologically or physiologically active agents, e.g., antigenicand/or other biologically active substances.

[0209] Formulations of the present invention can be presented, forexample, as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the vector deliverystructure; or as a suspension.

6. EXAMPLES

[0210] An isogenic series of expression plasmids composed of individualcassettes has been constructed for use in bacterial live vectorvaccines, such as E. coli and Salmonella. With the exception ofribosomal binding sites (RBS), the key genetic loci controllingtranscription initiation and termination, plasmid replication, orencoding expressed proteins are contained within defined restrictionfragments, as depicted by the representative plasmid diagram of pGEN2seen in FIG. 1A. The basic structure of these expression plasmids willfirst be highlighted and then the data demonstrating the function ofeach locus within the attenuated vaccine strain CVD908-htrA will besummarized.

[0211] 6.1 pGEN Structure

[0212] Transcription of any heterologous antigen to be expressed withinCVD908-htrA is primarily controlled by an inducible promoter containedon an EcoRI-BglII cassette. Since the expression plasmids were initiallymodeled after pTETnir15, early versions carried theanaerobically-activated nir15 promoter (P_(nir15)). However, thispromoter has been replaced with a more tightly regulated osmoticallycontrolled promoter P_(ompC) which is easily manipulated in vitro byvarying the concentration of NaCl.

[0213] Heterologous antigens are contained on a BglII-AvrII cassette,flanked by an optimized RBS at the 5′-proximal end and a trpAtranscriptional terminator at the 3′-distal end of this cassette. Theorigin of replication for these expression plasmids has been designed asan AvrII-BglII cassette, and is protected from read-throughtranscription originating in flanking regions. These cassettes carry anextremely efficient derivative of the T1T2 transcriptional terminator atone terminus with the trpA transcriptional terminator from theheterologous antigen cassette at the opposite end of the replicationcassette.

[0214] The flanking BglII and SpeI sites (see FIG. 2) between thereplication cassette and the selection cassette are intended forinsertion of a plasmid maintenance function, such as the par locus frompSC101. The selection cassettes contained within the plasmids arecontained within SpeI-XbaI cassettes, and can, for example, be used toencode resistance to carbenicillin (the bla gene) or resistance totetracycline (the tetA gene, see FIG. 1).

[0215] The drug resistance cassette can be replaced with the ssb geneencoding the essential single stranded binding protein of Salmonellatyphi CVD908-htrA.

[0216] The flanking XbaI and EcoRI sites between the selection cassetteand P_(ompC) are intended for insertion of additional maintenancefunctions, including a PSK locus such as hok-sok (see FIGS. 1 and 2), oran additional partition function such as the parA locus from pR1 (seeFIG. 7).

[0217] 6.2 Modified ompC Promoter

[0218] It was intended that any promoter controlling transcription of aheterologous gene be responsive to an environmental signal of biologicalrelevance. For the expression plasmids described here, an ompC promotercassette (P_(ompC)) from E. coli was used, which is induced by increasesin osmolarity. Construction of this cassette was based on the publishedsequence of P_(ompC) published by Norioka et al (Norioka et al. 1986)and was carried out using synthetic primers to create a 459 bpEcoRI-BglII cassette in which the natural RBS was removed.

[0219] To confirm that this promoter was osmotically controlled withinCVD 908-htrA, a derivative of pTETnir15 was constructed in whichP_(nir15)-toxC was replaced by a cassette comprised of P_(ompC) drivingexpression of a promoterless aphA-2 cassette conferring resistance tokanamycin. This plasmid, designated pKompC, was introduced into CVD908-htrA by electroporation, and recipients were screened for resistanceto kanamycin on LB medium. The osmotically regulated expression ofaphA-2 was determined by inoculating CVD 908-htrA(pKompC) into 50 ml ofsupplemented nutrient broth (NB) containing increasing concentrations ofkanamycin from 0 to 300 μg/ml; a parallel set of cultures were set upwith the identical ranges of kanamycin added, but also containing 10%sucrose to induce P_(ompC). Cultures were incubated overnight at 37° C.,and the O.D.₆₀₀ was measured. Results are reported in the Table 2,Experiment 1.

[0220] TABLE 2 shows induction with osmolarity of the promoter P_(ompC),controlling expression of resistance to kanamycin, within the attenuatedS. typhi live vector CVD 908-htrA. TABLE 2 EXPERIMENT 1¹ EXPERIMEMT 2²Concen- Concen- tration tration of Low 10% of Low 300 mM kanamycinosmolarity sucrose kanamycin osmolarity NaCl (μg/ml) (O.D.₆₀₀) (O.D.₆₀₀)(μg/ml) (O.D.₆₀₀) (O.D.₆₀₀) 0 0.92 0.35 0 0.95 1.04 50 0.13 0.35 2000.04 0.99 100 0.07 0.31 400 0.02 0.96 200 0.03 0.21 600 0.01 0.92 3000.02 0.19 800 0.01 0.92 # but with increasing concentrations ofkanamycin; a parallel set of cultures were set up with the identicalranges of kanamycin added, but also containing 10% sucrose to hopefullyinduce P_(ompC). Cultures were incubated overnight at 37° C., and theO.D.₆₀₀ was measured. # and all cultures were incubated at 37° C., for16 hr. and the O.D.₆₀₀ was measured.

[0221] Regardless of selective pressure using kanamycin, the presence of10% sucrose had an inhibitory effect on the growth of CVD908-htrA(pKompC). However, the results suggested that E. coli PompC wasosmotically controlled when driving aphA-2 gene expression within CVD908-htrA(pKompC). To confirm this, CVD 908-htrA(pKompC) was inoculatedinto 50 ml of supplemented NB broth, containing increasingconcentrations of kanamycin from 200 to 800 μg/ml; a parallel set ofcultures was again set up containing 300 mM NaCl to induce P_(ompC).Cultures were incubated at 37° C. for 16 hr, and results are reported inTable 2, Experiment 2. It was confirmed that PompC-driven expression ofthe aphA-2 gene within CVD 908-htrA confers resistance to kanamycin atlevels up to 800 μg/ml in an osmotically regulated manner.

[0222] The aph gene cassette was then replaced with a 756 bp BglII-NheIcassette containing the gfpuv allele encoding GFPuv. During the visualscreening of E. coli colonies sub-illuminated with ultraviolet light,one very brightly fluorescing colony and another representativefluorescent colony were chosen for further study, designated clone 1 andclone 3, respectively. Upon purification of the plasmids involved, itwas determined that clone 1 contained a plasmid that no longer carried aBglII site separating P_(ompC) and gfpuv, while clone 3 carried theexpected BglII site. We examined the induction of GFP expression whenclones 1 and 3 are grown on nutrient agar in the presence or absence ofNaCl, and determined by visual inspection that clone 3 displayed verylittle fluorescence when grown on nutrient agar containing no NaCl butfluoresced brightly when plated on nutrient agar containing 300 mM NaCl(data not shown). Clone 1, however, had a higher background level offluorescence when uninduced, but fluoresced intensely when induced with300 mM NaCl. To rule out mutations within the gfpuv gene which mightaffect fluorescence, we replaced P_(ompC) from clone 1 with P_(ompC)from clone 3, and confirmed the expected decrease in fluorescence asjudged by sub-illumination (data not shown). We therefore concluded thatdifferences in observed fluorescence were controlled by two geneticallydistinct versions of the P_(ompC) promoter, which we designate asP_(ompC1) (higher transcription levels with less osmotic control) andP_(ompC3) (moderate transcription levels with osmotic control similar tothat observed for the P_(ompC)-aph cassette described above); wedesignate the plasmids containing these expression cassettes aspGFPompC1 and pGFPompC3, respectively.

[0223] To quantify the differences in induced and uninduced expressionof gfpuv controlled by P_(ompC1) and P_(ompC3), GFPuv synthesis wasmonitored within both E. coli DH5α and S. typhi CVD 908-htrA using flowcytometry. This powerful technique has the unique advantages of allowingrapid measurement of GFPuv expression within large numbers of individualbacteria, as well as accurately determining the mean intensity offluorescence due to GFPuv synthesis within each bacterial populationanalyzed. To accomplish this, pGFPompC1 and pGFPompC3 were introduced byelectroporation, and colonies were isolated on supplemented 1× LB agarcontaining 100 μg/ml of carbenicillin grown at 30° C. for 48 hr.Isolated colonies were then grown up and cultures frozen down as masterstocks. Fresh colonies were then inoculated into either supplementednutrient broth or supplemented nutrient broth containing 150 mM NaCl,and grown at 37° C./250 rpm for 24 hr; the difference in O.D.₆₀₀ for anyculture was never greater than 0.07. Induction of expression of gfpuv,controlled by P_(ompC1) and P_(ompC3), was analyzed by flow cytometry,and results are presented in Table 3.

[0224] TABLE 3 shows a comparison of induction of P_(ompC1) andP_(ompC3), controlling expression of GFPuv, within the host strains E.coli DH5α and CVD 908-htrA.¹ TABLE 3 Mean Mean Low osmolarityFluorescence 150 nM Nacl Fluorescence Induction STRAIN (O.D.₆₀₀)Intensity (O.D.₆₀₀) Intensity Ratio² DH5α 0.61 0.28 0.95 0.29  NA³ DH5α0.56 4.45 0.72 7.69 1.7 (pGFPompC1) DH5α 0.58 1.77 0.73 4.21 2.4(pGFPompC3) CVD908-htrA 0.58 0.27 0.65 0.26 NA CVD908-htrA 0.60 5.370.54 23.4 4.4 (pGFPompC1) CVD908-htrA 0.54 2.56 0.53 17.1 6.7(pGFPompC3)

[0225] The basal level of expression for the P_(ompC1)-gfpuv cassette is2.5 times higher than for the P_(ompC3)-gfpuv cassette, when expressedin DH5α, and 2.1 times higher when expressed within CVD 908-htrA;however, the basal level of fluorescence detected for synthesis of GFPuvnever exceeded a mean fluorescent intensity of 5.37, regardless of hostbackground. If we define induction ratio as the ratio of meanfluorescent intensity measured after induction, divided by basal levelof mean fluorescent intensity, it was observed that when induced with150 mM NaCl, P_(ompC1) and P_(ompC3) displayed within DH5α inductionratios of 1.7 and 2.4 respectively. Surprisingly, the induction ratiofor P_(ompC1) when measured in CVD 908-htrA was 4.4, and produced amaximum mean fluorescence intensity of 23.4 for these experiments.Although the induction ratio for P_(ompC3) within CVD 908-htrA was 6.7,the mean fluorescence intensity of 17.1 was lower than measured forP_(ompC1). Based on these data, it appears that P_(ompC1) is thestrongest and yet osmotically controlled of the two ompC promoters.P_(ompC1) was therefore chosen for synthesis of the widest possiblerange of heterologous test antigen to examine the effects of suchsynthesis on plasmid stability.

[0226] These data clearly show that when driving expression of gfpuvwithin the live vector strain CVD 908-htrA, P_(ompC1) and P_(ompC3) areinducible with increasing osmolarity, although the basal level oftranscription is still noteworthy in both cases. The results observedunder conditions of low osmolarity further support our observationsusing solid media that P_(ompC1) drives higher heterologous antigenexpression than P_(ompC3). Since P_(ompC3) was noted to possess theintended 3′-terminal BglII site, which was not detected for P_(ompC1),we determined the nucleotide sequence for P_(ompC1) to perhaps detectpoint mutation(s) which might explain the strength of P_(ompC1). Theonly differences identified were located at the 3′-terminus of thecassette. The intended sequence within this region was 5′-. . .catataacAGATCTtaatcatccacAGGAGGatatctgATG-3′ (from left to right, uppercase denotes the BglII site, ribosome binding site, and GFPuv startcodon respectively); the actual sequence proved to be 5′- . . .catataacAGATCGATCTtaaAcatccacAGGAGGAtAtctgATG-3 (inserted or changedbases denoted with underlined bold upper case). These changes detectedwithin the ompC1 promoter sequence are apparently responsible forincreasing the observed strength of P_(ompC1) by an unknown mechanism,since neither the basic ompC promoter sequence, nor the optimizedribosome binding site have been spontaneously altered.

[0227] 6.3 Origins of Replication and Selection Cassettes

[0228] The success of expressing potentially toxic or otherwiseproblematic heterologous antigens within CVD908-htrA depends on the copynumber of the expression plasmid. In addition, observed immune responsesto a given heterologous antigen are affected by the copy number of thegene(s) encoding the antigen, with chromosomally expressed antigenseliciting poorer immune responses when compared to plasmid-basedexpression.

[0229] An optimized immune response will depend on multicopyplasmid-based expression of the heterologous antigen(s) from plasmidswith the appropriate copy number.

[0230] Since the appropriate copy number for a given heterologous genecannot be known a priori, the present invention provides a set ofexpression plasmids which contain the origins of replication oriE1(amplified from pAT153; copy number ˜60), ori15A (amplified frompACYC184; copy number 15), and ori101(amplified from pSC101; copy number˜5). These self-contained replication cassettes are all carried onBglII-BamHI fragments, and are depicted for a set of 3tetracycline-resistance expression plasmids shown in FIGS. 1A-1C.

[0231] Expression of the P_(ompC1)-controlled gfpuv expression cassettecontained on these expression plasmids was analyzed using flowcytometry. These experiments were designed to detect whether differencesin the level of observed fluorescence could be correlated with theexpected copy number of a given expression plasmid. CVD908-htrA strainscarrying pGEN2, pGEN3, and pGEN4 were streaked onto the rich mediumSuperAgar supplemented with DHB and 20 μg/ml tetracycline whereappropriate. SuperAgar was used because it is a very rich medium (3× LBagar). Plates were incubated at 30° C. to reduce the toxicity of GFPsynthesis and allow bacteria to grow luxuriously on the plates. Isolatedcolonies were then inoculated into 45 ml of SuperBroth supplemented withDHB and 20 μg/ml tetracycline where appropriate, and incubated at 37° C.for 16 hr. Bacteria were concentrated by centrifugation and resuspendedin 1 ml of sterile PBS, pH=7.4, and diluted 1:100 in PBS, pH=7.4 priorto FACS analysis. Bacteria were analyzed by flow cytometry, as describedabove, for two independent growth experiments, and results are displayedin Table 4 at the end of this section.

[0232] These data support the conclusion that overexpression of GFPuvwithin CVD908-htrA is toxic to the bacteria. As the theoretical copynumber increases for the plasmids pGEN4, pGEN3, and pGEN2 expressingGFPuv under identical growth conditions from the identical P_(ompC1)promoter, the percentage of the growing population which fluorescesdeclines. It is expected that the “dim” bacteria are not viable bacteriaand may no longer contain the expression plasmid, since these cultureswere grown in the presence of 20 μg/ml tetracycline. It is noted,however, that when streaked onto solid medium and grown at 37° C. for24-36 hr, CVD908-htrA(pGEN2) grows poorly and fails to produce isolatedcolonies, while CVD908-htrA(pGEN3) and CVD908-htrA(pGEN4) grow quitewell and produce intensely fluorescing isolated colonies.

[0233] GFPuv is employed herein as representative of other problematicheterologous antigens which would be of interest to include in abacterial live vector, such as the S. typhi-based live vector; however,it will be appreciated that GFPuv can be replaced by any non-metabolicprotein or peptide antigen.

[0234] The data above show that although use of medium-copy expressionplasmids containing oriE1 replicons can be of use in expression of someantigens, expression of antigens of higher toxicity will be moresuccessfully expressed from lower copy number plasmids which employorigins of replication yielding average copy numbers between 2 and 30,such as ori101 or ori15A origins of replication. TABLE 4 Experiment 1Experiment 2 Mean Mean Fluorescence Fluo- Of Dim Mean rescence PercentBacteria Percent Fluorescence Percent Of Percent Mean Dim (RelativeFluorescing (Relative Dim Dim Bacteria Fluorescing Fluorescence StrainBacteria Units) Bacteria Units) Bacteria (Relative Units) Bacteria(Relative Units) CVD908-htrA 100 0.6 0 0 100 0.3 0 0 CVD908- 19.9 0.180.1 38.5 37.2 0.3 62.8 10.1 htrA (pGEN2) CVD908- 17.1 0.1 82.9 28.1 4.90.2 95.1 8.28 htrA (pGEN3) CVD908- 12.1 0.1 88.0 22.4 9.4 0.3 90.6 4.25htrA (pGEN4)

[0235]6.4 The hok-sok Antisense Post-Segregational Killing Locus

[0236] Using the polymerase chain reaction, the hok-sok PSK genes wereamplified using the multiple antibiotic resistance R-plasmid pR1 as thetemplate in these reactions. All initial attempts to clone this locusonto either high or medium copy number plasmids were unsuccessful. Inorder to directly select for the hok-sok locus during subcloning, a setof primers was designed for use in overlapping PCR reactions such thatthe final product was a fragment containing a genetic fusion of thehok-sok locus from pR1 and a promoterless tetA gene from pBR322 encodingresistance to tetracycline. This cassette was engineered such thattranscription of the hok gene would continue into tetA; the two lociwithin this cassette were separated by an XbaI restriction site forfuture manipulations.

[0237] Construction of this cassette not only allowed for directselection of the hok-sok locus, but also allowed for confirmation thatthe PSK function would operate in S. typhi CVD908-htrA. Afterelectroporation of plasmids carrying the cassette into CVD908-htrA,transformants could be selected using tetracycline. Successful recoveryof isolated colonies indicates successful synthesis of the hok-tetAmRNA, and successful synthesis of the antisense sok RNA to preventtranslation and synthesis of Hok, which would kill the bacteria.Recovery of the hok-sok-tetA cassette then became straightforward, andwas easily incorporated into our expression plasmids to create theselectable marker cassette of the plasmids pGEN2, pGEN3, and pGEN4depicted in FIGS. 1A-1C.

[0238] Experiments were then initiated to determine the effect of thehok-sok PSK function on the stability of expression plasmids containingoriE1 and the resistance marker bla encoding β-lactamase which confersresistance to carbenicillin. The hok-sok cassette was inserted into thepAT153-based expression plasmid pTETnir15, in which the Pnir15-toxCheterologous antigen cassette was replaced with our P_(ompC1)-gfpuvcassette, creating the plasmids pJN72 (without hok-sok) and pJN51 (withhok-sok). An additional set of plasmids was created by replacingP_(ompC1) with the weaker promoter P_(ompC3), creating pJN10 and pJN12;the structures of these four isogenic plasmids are represented in FIG.2. CVD908-htrA strains carrying either pJN72, pJN51, pJN10, or pJN12were streaked onto the rich medium SuperAgar supplemented with DHB and100 μg/ml carbenicillin, and plates were incubated as above for the pGENplasmids at 30° C. to reduce the toxicity of GFPuv synthesis and allowbacteria to grow luxuriously on the plates.

[0239] Isolated colonies were then inoculated into 45 ml of Super brothsupplemented with DHB and 100 μμg/ml carbenicillin and grown at 37° C.for 24 hours for analysis by flow cytometry of fluorescence. A secondindependent experiment was carried out exactly as the first, exceptisolated colonies were suspended in 500 μl of Super broth and 250 μleach inoculated into 45 ml paired Super broth cultures with or without300 mM NaCl added to induce the P_(ompC)-gfpuv cassettes; cultures wereincubated at 37° C. for 48 hrs and again analyzed by flow cytometry; andresults for both experiments are displayed in Table 5. Fluorescencehistograms for uninduced and induced expression plasmids from experiment2 are represented in FIGS. 3A-3H. TABLE 5 Experiment 1 Experiment 2 MeanMean Fluorescence Fluo- Percent Of Percent Mean rescence % Mean Dim DimFluorescing Fluo- +/−300 mM % Dim Dim Fluorescing Fluo- Strain BacteriaBacteria Bacteria rescence O.D.₆₀₀ NaC1 Bacteria Bacteria Bacteriarescence CVD908- 100 0.3 0.73 − 100 0.3 0 0 htrA CVD908- 3.1 0.2 96.910.2 0.75 − 2.3 0.3 97.7 11.7 htrA (pJN72) 0.89 + 22.2 0.3 77.8 22.5CVD908- 58.1 0.3 41.9 6.29 0.62 − 56.3 0.3 43.7 18.4 htrA (pJN51) 0.82 +95.4 0.3 4.6 21.0 CVD908- 5.4 0.2 94.6 7.43 0.72 − 1.7 0.3 98.3 8.3 htrA(pJN10) 0.96 + 29.9 0.3 70.1 19.8 CVD908- 18.9 0.2 81.1 6.60 0.47 − 45.20.3 54.8 16.4 htrA (pJN12) 0.68 + 95.6 0.3 4.4 13.2

[0240] These flow cytometry results can be explained as follows:expression of GFPuv (or other potentially detrimental heterologousantigen) from a multicopy expression plasmid such as pJN72 increases themetabolic stress on the CVD 908-htrA(pJN72) live vector, and increasesplasmid instability in the absence of selection. Since the selectablemarker of the expression plasmid encodes the secreted enzymeβ-lactamase, then as time increases the concentration of carbenicillinin the surrounding medium declines, selective pressure decreases, andthe frequency of plasmid loss increases; however, since multicopyplasmids are involved, relatively few bacteria succeed in losing allresident plasmids, but the average copy number of pJN72 per bacteriumdrops.

[0241] Quantitation by flow cytometry of GFPuv production for anuninduced population of healthy growing CVD 908-htrA(pJN72) indicatesthat the majority of bacteria express GFPuv and few non-fluorescingcells are detected (FIG. 3A). However, increasing production of GFPuv byinduction of the P_(ompC1)-gfpuv cassette increases the metabolic stresson CVD 908-htrA(pJN72), and although the production of GFP doubles, thepercentage of non-fluorescent bacteria increases as more plasmids arelost from the population (FIG. 3B).

[0242] In a similar population of growing CVD 908-htrA(pJN51), eachbacterium carries multicopy plasmids encoding both GFPuv and a PSKfunction. The frequency of plasmid loss for pJN51 remains the same asfor pJN72, but in this case as individual bacteria lose copies of theexpression plasmid, the 1:1 stoichiometry between the mRNA levels of hokand sok is disturbed, and production of Hok leads to cell death;therefore, the only CVD 908-htrA(pJN51) bacteria that will grow rapidlywill be those which retain all of their expression plasmids.Accordingly, it is not surprising that quantitation by flow cytometry ofGFPuv production for an uninduced population of healthy growing CVD908-htrA(pJN51) now detects a population of fluorescing bacteria whichdisplays levels of GFPuv fluorescence equivalent to those observed forCVD 908-htrA(pJN72) grown under inducing conditions (FIG. 3C vs FIG.3B); however, the percentage of non-fluorescing bacteria rises to overhalf the overall population of organisms.

[0243] Increasing production of GFPuv in this population by induction ofthe P_(ompC1)-gfpuv cassette in CVD 908-htrA(pJN51) again increases themetabolic stress on the live vector, but now the percentage ofnon-fluorescent bacteria almost completely overtakes the few fluorescingbacteria as many plasmids are presumably lost from the population andbacteria are killed (FIG. 2D).

[0244] One would expect that if a weaker promoter is used to controlexpression of GFPuv, the overall fluorescence of the population would bedecreased (compared to that observed for a similar population oforganisms grown with a strong promoter expressing GFPuv under identicalconditions), and the percentage of non-fluorescent bacteria should dropdue to the overall drop in GFPuv synthesis. However, as seen in FIGS.3E-3H, use of the weaker P_(ompC3)-gfpuv cassette did not significantlyimprove the viability of induced bacteria carrying a killing system,even though overall expression of GFPuv was reduced.

[0245] It is concluded that in order to maximize the percentage of apopulation of live vectors expressing the heterologous antigen ofchoice, it is not sufficient only to incorporate a PSK function into agiven expression plasmid, whether it be a drug resistance marker, theasd system, an alternate ssb system, or the hok-sok killing system. Inaddition to optimizing copy number and expression levels, thesegregation frequencies of these plasmids must also be improved toensure that each daughter cell in an actively growing population willinherit at least one expression plasmid and those that do not will bekilled and removed from the population. It is therefore within the scopeof the present invention to provide an expression plasmid having a PSKfunction and further having optimized copy number and/or expressionlevels, coupled with incorporation of one or more SEG functions.

[0246] 6.5 Complementation-based Killing System

[0247] It is also within the broad scope of the present invention toprovide an expression plasmid comprising a complementation-based killingsystem, for example, a system involving the deletion of the chromosomalssb locus of CVD908-htrA by homologous recombination, andtrans-complementation of this lesion using multicopy plasmids carryingfunctional ssb.

[0248] To carry out such constructions requires cloning the relevantsection of the S. typhi chromosome encompassing the ssb gene andflanking sequences, into which specific deletions can be introduced forchromosomal mutagensis.

[0249] Since our original submission, substantial progress has been madein the sequenceing of the Salmonella typhi chromosome at the SangerCentre in London. The Sanger Centre is a genome research center set upin 1992 by the Wellcome Trust and the Medical Research Council in orderto further our knowledge of genomes. Among other projects, the SangerCentre is sequencing the 4.5 Mb genome of S. typhi, in collaborationwith Gordon Dougan of the Department of Biochemistry, Imperial College,London. They are sequencing strain CT18, a highly pathogenic, multipledrug resistant strain isolated from a typhoid patient in Cho QuanHospital, Ho Chi Minh City, Vietnam. This strain is known to harborpVN100 (a 130 kb multidrug resistance plasmid) and a cryptic 80 kbplasmid. The genome is being sequenced by a whole genome shot gunapproach using a 2 kb pUC library, generated inhouse from chromosomalDNA supplied by Prof. Dougan's lab. Each insert is being sequenced oncefrom each end. The shotgun phase is now complete, and finishing hasbegun. At present there are 60 contigs over 1 kb in the database; atotal of 5.106 Mb of sequence assembled from 87,331 reads.

[0250] Based on updated results posted Oct. 4, 1999, we have identifiedContig 343, which contains the S. typhi ssb locus and critical flankingsequences within a 205,199 bp region. We have designed primers 1 and 4(listed below) to amplify by PCR a 3535 bp fragment of the S. typhichromosome in which the ssb locus is flanked by 1.5 kb of chromosomalsequence; this flanking symmetry is required for optimal crossoverfrequenceis to introduce the counter-selectable sacB-neo cassette andreplace ssb. Using the methodology previously filed, we will use primers1 and 2 to engineer a 5′-proximal 1.5 kb Eco RI-Xma I cassette, upstreamof ssb. Primers 3 and 4 will be used to generate the 3′-distal 1.5 kbXma I-Eco RI cassette, downstream of ssb; both 1.5 kb cassettes will beligated together, forming the 3 kb Eco RI fragment containing a uniqueXma I site exactly in the middle of the cassette. The sacB-neo cassettecan now easily be inserted into the Xma I site, to complete constructionof the mutagenesis cassette to be inserted into pCON (previouslydescribed in our first filing). The required complementing ssb-1cassette will be constructed using primers 5 and 6 as a Nhe I cassettefor replacement of drug resistance markers within the Xba I-Spe Icassettes of pGEN 211, pGEN 222, pGEN 206, or any later version of theexpression plasmids detailed herein. PRIMER 1:5′-gaattcGCGCGCTTCGCGATTCAGTCGCGTTCCTTCACAGCTGGCGCAGGGGCGATTACTGATGAA-3′ PRIMER 2:5′-cccggGAGTCTCCTGAATACGTTTCATAAATAGTGTAAACGCGTGAGTGTACCATTTCCACGTAGC-3′ PRIMER 3:5′-cccggGTAAAAAACTCAAAGCGTTATTTGCATTTTCGCTATAGTTCTCGTCTGCTGAAATGCCTGGTGT-3′ PRIMER 4:5′-gaattcCATTTCTATCAATAAATTACTATTAGTTTTGTCTTCTAACCAAGCCTCTATTTTATGAGTATCCTCTTCAG-3′ PRIMER 5:5′-gctagcATGGCCAGCAGAGGCGTAAACAAGGTGATTCTCGTTGGTAATCTGGGCCAGGACCCGGAAGTACGC-3′ PRIMER 6:5′-gctagcTCAGAACGGAATGTCGTCGTCAAAATCCATTGGC GGTTCGTTAGACGGCGCTGGCGCG-3′

[0251] 6.6 Stability of Expression Plasmids in the Absence of Selection

[0252] In order to develop a non-catalytic plasmid maintenance system toenhance the stability of multicopy expression plasmids encoding foreignantigens within CVD 908-htrA, experiments were initiated to monitorplasmid stability by quantitating expression of GFPuv by flow cytometrywhen strains were passaged in the absence of antibiotic selection. Theseexperiments were designed to address 3 fundamental questions: 1] What isthe effect of the induction level of P_(ompC1) on the stability ofplasmids encoding a heterologous antigen such as GFPuv? 2] What is theeffect of copy number on the stability of plasmids expressing GFPuv? 3]How do the hok-sok, par, and parA maintenance functions affect plasmidretention, both as individual components and synergistically?

[0253] Initial flow cytometry experiments were carried out in which CVD908-htrA carried replicons with either the oriE1, ori15A, or ori101origin of replication. It was quickly determined that replicons carryingthe higher copy number oriE1 origins were very unstable, even whenstrains were grown in the presence of antibiotic selection. Flowcytometry results indicated that even when cultured in the presence ofcarbenicillin, the percentage of the bacterial populations no longerexpressing detectable GFPuv ranged from approximately 50% for pGEN71(carrying hok-sok) and pGEN84 (hok-sok+par) to 62% for pGEN211(hok-sok+par+parA). Since replicons carrying an oriE1 origin clearly didnot allow for optimal synthesis of the heterologous GFPuv test antigenwithin the majority of a growing population of live vector bacteria,this series of expression plasmids was not examined further.

[0254] CVD 908-htrA carrying expression plasmids with an ori15A originwere then examined. Strains were inoculated into 25 ml cultures of1×LB+DHB (no antibiotic selection) containing either 50 mM, 150 mM, or300 mM NaCl. Cultures were incubated for 24 hr at 37 C./250 rpm, diluted1:1000 into fresh medium of identical osmolarity, and incubated foranother 24 hr; samples from all cultures were analyzed for levels ofGFPuv synthesis by flow cytometry. Results for the first passage in theabsence of selection are listed in Table 6, and the histogramsrepresenting these data are shown in FIG. 8.

[0255] TABLE 6 shows stability within CVD 908-htrA of ori15A replicons,containing plasmid maintenance systems of increasing complexity, grownwithout selection and in the presence of increasing osmolarity.¹ TABLE 650 mM NaCl 150 mM NaCl 300 mM NaCl Percent Mean Percent Mean PercentMean Fluorescing Fluorescence Fluorescing Fluorescence FluorescingFluorescence STRAIN² O.D.₆₀₀ Bacteria Intensity O.D.₆₀₀ BacteriaIntensity O.D.₆₀₀ Bacteria Intensity CVD908-htrA 0.98 100 0.6 1.11 1000.6 1.12 100 0.6 pGEN91 1.00 13.2 28.6 1.17 11.4 42.9 1.26 10.9 65.5pGEN111 1.26 47.4 51.8 1.17 28.9 93.6 1.12 42.4 65.1 pGEN121 1.01 80.553.3 1.20 73.8 74.0 1.15 56.7 105.3 pGEN193 1.11 71.4 50.9 1.24 65.264.7 1.22 53.7 90.8 pGEN222 1.01 96.8 52.1 1.28 93.3 67.8 1.13 95.3 89.2# this table, bacteria were then pelleted, resuspended in 1 ml PBS pH7.4, and then diluted 1:1000 into PBS for analysis by flow cytometry.

[0256] In general, as osmolarity increases and induction of P_(ompC1)rises, the percentage of the live vector population expressing GFPuvdrops; nevertheless, the mean level of fluorescence intensity increasesas expected. For example, in the presence of 50 mM NaCl, 80.5% of apopulation of CVD 908-htrA(pGEN121) express GFPuv with a meanfluorescence intensity of 53.3. As the concentration of NaCl increasesto 300 mM NaCl, the percentage of the population expressing GFPuv dropsto 56.7%; nevertheless, the mean fluorescence intensity rises to 105.3.However, it is notable that for strains carrying pGEN222 with a completeplasmid maintenance system (i.e hok-sok+par+parA), the percentage of thepopulation expressing the heterologous antigen remains at approximately95%, while the mean fluorescence intensity increases from 52.1 (50 mMNaCl) to 89.2 (300 mM NaCl). It was noted that upon further passage ofthese strains for an additional 24 hrs in the absence of antibioticselection, less than 5% of bacteria continued to express functionalGFPuv. Streaks of these cultures onto solid medium, prior to flowanalysis, indicated that non-fluorescing bacteria remained viable, butwere sensitive to antibiotic selection. When non-fluorescing bacteriawere sorted and plated, they were confirmed to be sensitive toantibiotic and non-fluorescent when irradiated with ultraviolet light,indicating loss of resident plasmids.

[0257] A passage experiment involving CVD 908-htrA carrying expressionplasmids with an ori101 origin detected no significant loss of GFPuvexpression after passage of strains for 48 hrs without selection,regardless of osmolarity. Therefore, strains were passaged in a separateexperiment for 96 hrs (i.e. 4×24 hr) in the presence of either 50, 150,or 300 mM NaCl. Populations were analyzed by flow cytometry after 3 and4 passages, and results are recorded in Table 7.

[0258] TABLE 7 shows stability within CVD 908-htrA of ori101 replicons,containing plasmid maintenance systems of increasing complexity, grownwithout selection and in the presence of increasing osmolarity. TABLE 750 mM NaCl 150 mM NaCl 300 mM NaCl STRAIN Percent Mean Percent MeanPercent Mean (Passage Fluorescing Fluorescence Fluorescing FluorescenceFluorescing Fluorescence Number)¹ O.D.₆₀₀ Bacteria Intensity O.D.₆₀₀Bacteria Intensity O.D.₆₀₀ Bacteria Intensity CVD908-htrA (#3) ND² 1000.6 ND 100 0.5 ND 100 0.5 CVD908-htrA (#4) 1.00 100 0.3 1.18 100 0.31.19 100 0.3 pGEN132 (#3) ND  45.5 29.0 ND 33.2 36.9 ND 81.3 47.3pGEN132 (#4) 1.03 10.9 27.8 1.20 7.6 36.1 1.32 51.3 47.5 pGEN142 (#3)1.05 99.5 35.5 1.23 98.9 45.1 1.28 96.5 47.8 pGEN142 (#4) 1.17 94.4 38.01.29 91.5 45.0 1.33 93.9 47.7 pGEN206 (#3) 1.08 98.1 36.2 1.25 94.5 42.81.29 95.2 47.4 pGEN206 (#4) 1.13 80.2 32.6 1.26 68.6 36.6 1.33 93.5 41.3# For passage #2, 25 μl from passage #1 were inoculated into 25 ml (i.e.1:1000 dilution) of identical medium and incubated at 37° C., 250 rpmfor an additional 24 hr without selection. Passages 3 and 4 were carriedout in identical fashion, but after the next passage had been set up theremaining bacteria were then pelleted, resuspended in 1 ml PBS pH 7.4,and then diluted 1:1000 into PBS for analysis by flow cytometry.

[0259] Live vectors carrying unstabilized ori101 replicons eventuallylost the capacity to synthesize the heterologous antigen after 96 hr.For example, after 96 hr growth in the presence of 50 mM NaCl, only10.9% of CVD 908-htrA(pGEN132) expressed GFPuv and fluoresced. As theconcentration of NaCl in the medium was increased to 150 mM,fluorescence was detected in only 7.6% of the population; curiously, at300 mM NaCl, the percentage recovered to 51.3% fluorescing bacteria.Remarkably, CVD 908-htrA carrying either pGEN142 (hok-sok) or pGEN206(hok-sok+parA) retained synthesis of GFPuv in greater than 95% of thepopulation after 3 passages (72 hr), regardless of osmolarity (see Table7). The percentage of fluorescing CVD 908-htrA (pGEN142) remained nearthis level after 4 passages (96 hr), while decreasing slightly for CVD908-htrA (pGEN206).

[0260] Taken together, these data show that as copy number is reduced,the apparent stability of resident plasmids and proficiency of a livevector to synthesize a heterologous antigen such as GFPuv increases; asplasmid maintenance systems accumulate within a given plasmid, apparentstability and antigen synthesis are further enhanced. In addition, asthe induction of P_(ompC1) and concomitant production of theheterologous antigen increases, the percentage of a growing populationremaining capable of synthesizing antigen can be dramatically reduced.

[0261] 6.7 Bacterial Strains and Culture Conditions

[0262] All plasmid constructions were recovered in Escherichia colistrain DH5α or DH5αF′IQ (Gibco BRL). Construction of the hok-sok genecassette used pR1 template DNA isolated from E. coli strain J53(pR1), agenerous gift from James B. Kaper. The live vector S. typhi CVD 908-htrAis an auxotrophic derivative of the wild type strain Ty2 with deletionsin aroC, aroD, and htrA (Tacket et al. 1997b). All strains used forexamination of plasmid stability were grown in media supplemented with2,3-dihydroxybenzoic acid (DHB) as previously described (Hone et al.1991; Galen et al. 1997). When grown on solid medium, plasmid-bearingstrains of CVD 908-htrA were streaked from frozen (−70° C.) masterstocks onto 2× Luria-Bertani agar containing (per liter) 20 g Bactotryptone, 10 g Bacto yeast extract, and 3 g NaCl (2× LB agar) pluscarbenicillin at a concentration of 50 μg/ml. Plates were incubated at30° C. for 24-36 hr to obtain isolated colonies ˜2mm in diameter;strains were incubated at 30° C. to minimize the toxicity of GFPuvexpression in CVD 908-htrA.

[0263] When grown in liquid medium, cultures were incubated at 37° C.,250 rpm for 16-24 hr. To examine the osmotic induction of the ompCpromoter (P_(ompC)) within either E. coli DH5α or CVD 908-htrA, strainswere grown in Bacto nutrient broth (Difco) containing DHB and eitherNaCl or sucrose; cultures were supplemented either with 50 μg/ml ofcarbenicillin or increasing concentrations of kanamycin whereP_(ompC)-aphA-2 cassettes were examined. For quantitation of GFPuvsynthesis using flow cytometry, 6-8 isolated colonies from master stocksstreaked onto 2× LB agar as above were inoculated into 25 ml of 1× LBbroth supplemented with 50 μg/ml carbenicillin where desired and NaCl atincreasing concentrations to increase the induction of ompC promoters.Cultures were incubated at 37° C., 250 rpm for 16-24 hr prior topelleting bacteria for flow cytometry as described below.

[0264] 6.8 Molecular Genetic Techniques.

[0265] Standard techniques were used for the construction of theplasmids represented here (Sambrook et al., 1989). Unless otherwisenoted, native Taq DNA polymerase (Gibco BRL) was used in polymerasechain reactions (PCR). S. typhi was prepared for electroporation ofrecombinant plasmids after harvesting from Miller's LB broth (Gibco BRL)supplemented with DHB; after pelleting bacteria, the cells were washedthrice with one culture volume of sterile distilled water andresuspended in sterile distilled water to a final volume of {fraction(1/100)} of the original culture volume. Electroporation of strains wasperformed in a Gene Pulser apparatus (Bio-Rad) set at 2.5 kV, 200Ω, and25 μF. Following electroporation, bacteria were repaired using SOCmedium and incubating at 37° C., 250 rpm for 45 min; bacteria were thenplated on 1× LB medium containing DHB plus 50 μg/ml carbenicillin, andincubated at 30° C. for 24 hr. Isolated colonies were then swabbed ontosupplemented 2× LB and incubated at 30° C. for 16 hr. Frozen masterstocks were prepared by harvesting bacteria into SOC medium withoutfurther supplementation and freezing at −70° C.

[0266] 6.9 Construction of Expression Vectors

[0267] The expression vectors listed in the following Table 8 wereprepared in the course of the recent work. TABLE 8 Size Plasmid (kb)Relevant genotype Reference pTETnir15 3.7 oriE1 toxC bla Oxer et al.(1991) pJN1 1.9 oriE1 bla This work pJN2 3.4 oriE1 toxC bla This workpGFPuv 3.3 pUC19ori gfpuv bla Clontech pGFPompC 3.5 oriE1 gfpuv bla Thiswork pNRB1 3.5 oriE1 gfpuv tetA This work pGEN2 4.2 oriE1 gfpuv tetAhok-sok This work pGEN3 4.1 ori15A gfpuv tetA hok-sok This work pGEN45.6 ori101 gfpuv tetA hok-sok This work pJN5 3.1 oriE1 gfpuv bla Thiswork pJN6 3.7 oriE1 gfpuv bla hok-sok This work pJN7 4.1 oriE1 gfpuv blahok-sok par This work pJN8 5.4 oriE1 gfpuv bla hok-sok parA This workpGEN51 3.6 oriE1 gfpuv bla This work pGEN71 4.2 oriE1 gfpuv bla hok-sokThis work pGEN84 4.5 oriE1 gfpuv bla hok-sok par This work pGEN183 5.9oriE1 gfpuv bla hok-sok parA This work pGEN211 6.2 oriE1 gfpuv blahok-sok par parA This work pGEN91 3.5 ori15A gfpuv bla This work pGEN1114.1 ori15A gfpuv bla hok-sok This work pGEN121 4.5 ori15A gfpuv blahok-sok par This work pGEN193 5.8 ori15A gfpuv bla hok-sok parA Thiswork pGEN222 6.2 ori15A gfpuv bla hok-sok par parA This work pGEN132 4.8ori101 gfpuv bla par This work pGEN142 5.4 ori101 gfpuv bla par hok-sokThis work pGEN206 7.1 ori101 gfpuv bla par hok-sok parA This work

[0268] 6.9.1 Construction of pJN1 and pJN2

[0269] The expression plasmids constructed for these studies arecomposed of 3 basic cassettes encoding 1] expression of a heterologousantigen, 2] a plasmid origin of replication, and 3] selection andmaintenance functions. To accomplish this, a basic replicon wasconstructed in which these cassettes were separated by uniquerestriction sites. The primers used in construction of the plasmidcassettes are set forth in the following Table 9: TABLE 9 GenBank PrimerCassette Accession Region of Region of Number Sequence¹ created NumberHomology² Complementarity³  1 5′-GCAGGAAAGAACATGTGAGCCTA onE1 J017492463-2507 GGGCCAGCAAAAGGCCAGGAAC-3′  2 5′-CATGACCAAAATCCCTTAACTAG ″ ″3197-3145 TGTTTTAGATCTACTGAGCGTCAGAC CCCG-3′  35′-CGGGGTCTGACGCTCAGTAGATC bla ″ 3145-3197 TAAAACACTAGTTAAGGGATTTTGGTCATG-3′  4 5′-GCTGTCAAACATGAGAATTCTAG ″ ″ 17-1,AAGACGAAAGGGCCTCGTGATACGC 4361-4330 C-3′  5 5′-ACAGCCTGCAGACAGATCTTGACaphA-2 V00618  1-64 AGCTGGATCGCACTCTGGTATAATTG GG AAGCCCTGCAAAG-3′  65′-CGAAGCCCAACCTTTCATAGAAG ″ ″ 1044-986  CTAGCGGTGGATCCGAAATCTCGTGA TGGCAGGTTG-3′  7 5′-AACAAGCGTTATAGGAATTCTGT P_(ompC) K00541  4-33GGTAGCA-3′  8 5′-ACTTTCATGTTATTAAAGATCTG ″ ″ 498-469 TTATATG-3′  95′-AGATCTTAATCATCCAGAGGAGG gfpuv U62636 289-317CTTTCTGATGAGTAAAGGAGAAGAAC TTTTCACTGG-3′ 10 5′-GCTAGCTCATTATTTGTAGAGCT ″″ 1008-983  CATCCATGC-3 11 5′-AGATCTGAATTCTAGATCATGTT tetA J01749  4-41TGACAGCTTATCATCGATAAGCTTTA ATGCG-3′ 12 5′-AGATCTTATCAGGTCGAGGTGGC ″ ″1275-1234 CCGGCTCCATGCACGGCGACGCAACG CG-3′ 13 5′-CGCGAATTCTCGAGACAAACTCChok-sok- X05813  2-48 GGGAGGCAGCGTGATGCGGCAACAA tetA TCACACGGATTTC-3′ 145′-ATGAGCGCATTGTTAGATTTCAT ″ J01749, 108-86, TTTTTTTCCTCCTTATTTTCTAGACAX05813 580-559 A CATCAGCAAGGAGAAAGG-3′ 15 5′-CCTTTCTCCTTGCTGATGTTGTC ″X05813,  559-580, TAGAAAATAAGGAGGAAAAAAAAATG J01749  86-108AAATCTAACAATGCGCTCAT-3′ 16 5′-GCTACATTTGAAGAGATAAATTG ori15A X064031461-1397 CACTGGATCCTAGAAATATTTTATCT GATTAATAAGATGATC-3′ 175′-CGGAGATTTCCTGGAAGATGCCT ″ ″ 780-829 AGGAGATACTTAACAGGGAAGTGAGA G-3′18 5′-GTCTGCCGGATTGCTTATCCTGG ori101 X01654 4490-4550CGGATCCGGTTGACAGTAAGACGGGT AAGCCTGTTGAT-3′ 19 5′-CCTAGGTTTCACCTGTTCTATTA″ ″ 6464-6408 GGTGTTACATGCTGTTCATCTGTTAC ATTGTCGATCTG-3′ 205′-AGGCTTAAGTAGCACCCTCGCAA par X01654 4918-4858GATCTGGCAAATCGCTGAATATTCCT TTTGTCTCCGAC-3′ 21 5′-GAGGGCGCCCCAGCTGGCAATTCaphA2-pa V00618,  38-16, TAGACTCGAGCACTTTTGTTACCCGC rA X04268  1-37CAAACAAAACCCAAAAACAAC-3′ 22 5′-AGAAGAAAAATCGAATTCCAGCA ″ X042681704-1644 TGAAGAGTTTCAGAAAATGACAGAGC GTGAGCAAGTGC-3′ 235′-CGAAGCCCAACCTTTCATAGAAA ″ V00618 1044-986  CTAGTGGTGGAATCGAAATCTCGTGATGGCAGGTTG-3′ 24 5′-GTTGTTTTTGGGTTTTGTTTGGC ″ X04268, 37-1,GGGTAACAAAAGTGCTCGAGTCTAGA V00618 16-38 ATTGCCAGCTGGGGCGCCCTC-3′

[0270] pTETnir15 (see Table 8; Oxer et al. 1991) was re-engineered suchthat the oriE1 origin of replication and bla gene were separated by aunique SpeI site. Toward this end, an oriE1 cassette was synthesized byPCR using Vent polymerase with primers 1 and 2 and pCVD315 (Galen et al.1990) as the template. The resulting 735 bp fragment carries engineeredSpeI and BglII sites 5′-proximal to the promoter controllingtranscription of RNA II, and an engineered AvrII 675 bases from thesesites. A separate PCR reaction was carried out using primers 3 and 4 tocreate a 1234 bp bla cassette containing an engineered XbaI site5′-proximal to the original EcoRI site. The products from these two PCRreactions were gel purified and used in an overlapping PCR with primers1 and 4 to yield a final 1916 bp oriE1-bla fragment which wasself-ligated to create pJN1. The P_(nir15)-toxC fragment from pTETnir15was excised as an Eco RI (partial digestion)-AvaI fragment, in which theAvaI terminus was polished, and inserted into the multiple cloningregion from pSL1180 (Brosius, 1989) cleaved with Eco RI and StuI; thiscassette was then re-excised as an Eco RI (partial digestion)-AvrIIfragment and inserted into pJN1 leaved with Eco RI-AvrII, creating pJN2(see Table 8).

[0271] 6.9.2 Construction of pGFPompC

[0272] To facilitate screening of a functional osmotically regulatedP_(ompC) allele from Escherichia coli, an aphA-2 cassette wasconstructed, encoding resistance to the aminoglycosides neomycin andkanamycin (Shaw et al. 1993). A polymerase chain reaction (PCR) wascarried out using primers 5 and 6 with the template pIB279 (Blomfield etal. 1991) to generate a 1044 bp product, from which a promoterless 903bp aphA-2 BglII-NheI fragment was cleaved for replacement of aBglII-NheI toxC cassette encoding fragment C of tetanus toxin inpTETnir15. The anaerobically regulated P_(nir15) promoter was replacedwith a 459 bp EcoRI-BglII P_(ompC) allele constructed using primers 7and 8 with chromosomal template DNA from E. coli DH5α to create pKompC.After confirming osmotic induction of P_(ompC) by examining the increasein resistance to kanamycin with increasing osmolarity, the aphA-2cassette was then replaced with a gfpuv gene encoding a prokaryoticcodon-optimized GFPuv allele (Clontech; Crameri et al. 1996). The gfpuvgene was recovered by PCR using primers 9 and 10 with the templatepGFPuv to generate a 751 bp BglII-NheI fragment which was inserted intopKompC, to generate pGFPompC. Colonies were screened for functionalGFPuv, and the brightest colonies were then examined for induction offluorescence with increasing concentrations of NaCl. A P_(ompC1)-gfpuvcassette was cleaved from pGFPompC1 as an EcoRI-NheI fragment andinserted into a derivative of pJN2 cleaved with EcoRI-NheI to createpJJ4.

[0273]6.9.3 Construction of pNRB1, pGEN2, pGEN3, and pGEN4

[0274] Since it was intended that copy number not be influenced bytranscription originating from promoters outside the origin ofreplication, it was necessary to ensure that all replication cassetteswere flanked at both ends by transcription terminators. Because theorigin and antigen cassettes of pJN2 are separated by the trpAterminator, it was only necessary to insert one additional terminatorbetween the origin and bla cassettes.

[0275] To facilitate construction of additional plasmids later on, atetA-T1T2 cassette was created. pYA292 (Galan et al. 1990) was firstcleaved with HindIII and BglII, and the T1T2 terminator fragment waspolished and inserted into the SmaI site of the pBluescript 11 KS(Stratagene) mutiple cloning region; when the proper orientation wasidentified, this cassette was re-excised as a BamHI-PstI fragment andinserted into pIB307 (Blomfield et al. 1991) cleaved with BamHI-PstI,creating pJG14. It was later determined by sequence analysis that thecassette had undergone a deletion of approximately 100 bp, removing halfof the T2 terminator.

[0276] Using pBR322 as a template, primers 11 and 12 were used tosynthesize a 1291 bp tetA BglII fragment. This tetA BglII fragment wasthen inserted into the BamHI site of pJG14 such that transcription ofthe tetA gene is terminated at the T1T2 terminator, creating pJG14tetA.Finally, this tetA-T1T2 cassette was cleaved from pJG14tetA as anEcoRI-PstI fragment in which the PstI site had been removed bypolishing; the resulting fragment was inserted into pJJ4, cleaved withSpeI, polished, and recleaved with EcoRI to replace the bla cassette andcreate pNRB1.

[0277] The non-catalytic post-segregational killing function to beincorporated into the plasmid maintenance systems of the expressionplasmids described here was the hok-sok locus, from the multiple drugresistance R-factor pR1. Initial attempts at recovering the hok-soklocus after PCR were unsuccessful. It was therefore necessary to useoverlapping PCR to generate a cassette in which hok-sok wastranscriptionally fused to a promoterless tetA gene such thattranscription originating from the hok promoter would continue into tetAand result in a transcript encoding both Hok and resistance totetracycline. pR1 plasmid DNA was purified from E. coli J53(pR1) inwhich pR1 encodes resistance to both carbenicillin and chloramphenicol.A 640 bp hok-sok fragment was synthesized using primers 13 and 14; apromoterless 1245 bp tetA fragment was recovered in a separate PCR usingprimers 15 and 12 with pNRB1 as the template. The products from thesetwo PCR reactions were then used in an overlapping PCR with primers 12and 13 to yield the final 1816 bp hok-sok-tetA fragment. This fragmentwas inserted as an EcoRI-SphI fragment into pNRB1 cleaved withEcoRI-SphI, regenerating the tetA gene and creating pGEN1.

[0278] A set of 3 isogenic plasmids was then constructed, differing onlyin copy number, from which all further expression plasmids would bederived. The BglII-AvrII origin of replication cassette of pGEN1 wasreplaced by a BglII-AvrII oriE1 cassette from pJN2 to generate pGEN2. Anori15A replication cassette was synthesized by PCR using primers 16 and17 with pACYC184 template to generate a 629 bp BamHI-AvrII fragment,which was inserted into pGEN2 cleaved with BglII-AvrII to create pGEN3.Finally, an ori101 replication cassette was synthesized by PCR usingprimers 18 and 19 with pSC101 template, generating a 1949 bp BamHI-AvrIIfragment which was inserted into pGEN2 cleaved with BglII-AvrII tocreate pGEN4.

[0279] 6.9.4 Construction of pJN5, pGEN51, pGEN91, and pGEN132

[0280] The principle set of isogenic expression plasmids, to whichindividual elements of a plasmid maintenance system were sequentiallyadded, was composed of pGEN51 (containing oriE1), pGEN91 (containingori15A), and pGEN132 (containing ori101). The basic replicon from whichthese 3 plasmids were constructed was pJN5, which was assembled bycleaving the P_(ompC)-gfpuv cartridge as an EcoRI-NheI fragment frompGFPompC to replace the P_(nir15)-toxC cassette of pJN2. Construction ofpGEN51 was then accomplished by removal of the replication cassette frompGEN2 as a BamHI fragment, and replacement of the origin of replicationwithin pJN5 digested with BglII and BamHI, thereby regenerating thegfpuv gene. Construction of pGEN91 and pGEN132 were constructed in anidentical manner by excision of origin cassettes as BamHI fragments frompGEN3 and pGEN4 respectively (see FIG. 7 for representation of isogenicexpression plasmids based on pGEN91).

[0281] 6.9.5 Construction of pJN6, pGEN71, pGEN11I, and pGEN142

[0282] The hok-sok locus was then inserted as an XbaI-SalI fragment intopJN5 cleaved with XbaI and SalI, again regenerating the gfpuv gene tocreate pJN6 (see Table 2). Construction of pGEN71, pGEN111, and pGEN142was then carried out exactly as for pGEN51, pGEN91, and pGEN132 byinsertion into pJN6 of origin cassettes as BamHI fragments from pGEN 2,pGEN3, and pGEN4 respectively.

[0283] 6.9.6 Construction of pJN7, pGEN84, and pGEN121

[0284] Construction of oriE1 and ori15A expression plasmids containing aplasmid maintenance system, composed of both a post-segregationalkilling system and at least one partition function, was first attemptedusing the par function from pSC101. A 377 bp BamHI-BglII fragment wassynthesized using primers 18 and 20 with pSC101 template DNA; thisfragment was inserted into pJN6 cleaved with BglII to create pJN7. As inthe constructions above, origin cassettes from pGEN2 and pGEN3 were thenexcised as BamHI fragments and inserted into pJN7 digested with BglIIand BamHI to create pGEN84 and pGEN121.

[0285] 6.9.7 Construction of pJN8, pGEN183, pGEN193, pGEN206, pGEN211and pGEN222

[0286] The final expression plasmids were constructed by introduction ofthe parA active partitioning locus from pR1. As with hok-sok, initialattempts at recovering the parA locus after PCR were unsuccessful. Itwas necessary to use overlapping PCR to generate an aph-parA cassette,in which aph and parA were divergently transcribed and separated by XbaI and XhoI sites, to enable subcloning of the parA locus. A 1737 bp parAfragment was synthesized using primers 21 and 22 with pR1 template; a1076 bp aphA-2 fragment was recovered in a separate PCR using primers 23and 24 with pIB279 as the template. The products from these two PCRreactions were then used in an overlapping PCR with primers 22 and 23 toyield the final 2743 bp aphA2-parA fragment. This fragment was insertedas a 2703 EcoRI-SpeI fragment into pJN6. The parA cassette was thenre-excised as an XhoI fragment and inserted again into pJN6 cleaved withXhoI, regenerating the gfpuv gene, and creating pJN8.

[0287] Plasmids carrying a plasmid maintenance system composed of thepost-segregational killing hok-sok function and parA, were constructedby excision of oriE1 and ori15A BamHI-SpeI cassettes from pGEN51 andpGEN91 respectively, and insertion into pJN8 cleaved with BamHI and SpeIto create pGEN183 and pGEN193 respectively. Plasmids containing the fullcomplement of hok-sok, par, and parA maintenance functions wereconstructed by insertion of par-containing origin cassettes asBamHI-SpeI cassettes from pGEN84, pGEN121, and pGEN132 into pJN8 cleavedwith BamHI and SpeI to create pGEN211, pGEN222, and pGEN206respectively.

[0288] 6.10 Quantitation of GFPuv and Plasmid Maintenance

[0289] Quantitation of GFPuv and plasmid maintenance were analyzed bymeasuring the fluorescence of plasmid-bearing live vectors using anEpics Elite ESP flow cytometer/cell sorter system (Coulter) with theargon laser exciting bacteria at 488 nm and emissions detected at 525nm. 25 ml 1× LB cultures grown as described above were pelleted, andbacteria were resuspended into 1 ml of PBS. Cells were then diluted1:1000 into PBS prior to determination of viable counts and flowanalysis. Forward versus side light scatter, measured with logarithmicamplifiers, was used to gate on bacteria. A minimum of 50,000 eventswere acquired from each sample at a collection rate of approximately3500 events per second. Mean fluorescence intensity for a givenbacterial population was determined using the Epics Elite SoftwareAnalysis Package. The levels of autofluorescence, determined usingplasmidless S. typhi CVD 908-htrA and E. coli DH5a strains, were used toplace markers quantitating the percentages of bacteria in a givenpopulation expressing GFPuv.

[0290] 6.11 Conclusions

[0291] The broad objective of the research presented in Sections6.6-6.10 was to investigate the feasibility of developing a plasmidmaintenance system for the stabilization of multicopy expressionplasmids encoding foreign antigens in an S. typhi live vector vaccinestrain, without additional modification of the chromosome. Themaintenance of expression plasmids was enhanced at two independentlevels. First, dependence upon balanced-lethal maintenance systems thatinvolve catalytic enzymes expressed from multicopy plasmids was removed;this was accomplished through incorporation into expression plasmids ofa post-segregational killing system based on the non-catalytic hok-sokplasmid addiction system from the antibiotic-resistance factor pR1. Atleast one naturally occurring plasmid partition function was alsointroduced into these expression plasmids, to potentially eliminaterandom segregation of such plasmids, thereby enhancing their inheritanceand stability.

[0292] Although these expression plasmids are ultimately intended toexpress immunogenic and protective antigens for delivery to the humanimmune system, GFPuv was selected as a test reporter antigen becausequantitation of mean fluorescence in a population of growing livevectors could be used as a measure of the stability of resident plasmidswithin the live vector. All expression plasmids carried an identicalantigen expression cassette, with a P_(ompC1) allele controllingtranscription, and translation optimized by incorporation of a consensusribosome binding site. Because no catalytic activity is associated withthe fluorescence of GFPuv, the level of fluorescence intensity measuredby flow cytometry within individual bacteria could be correlateddirectly with gene dosage and copy number. In addition, use of anosmotically regulated ompC promoter allowed an assessment of plasmidstability and live vector viability as increasing osmolarity inducedhigher levels of GFPuv synthesis and presumably higher levels ofmetabolic stress on the live vector. As seen in Table 2, we confirmedthat the P_(ompC1) allele engineered for these studies was responsive toincreased osmolarity; when driving expression of an aph-2 resistancegene, resistance to less than 50 μg/ml kanamycin was observed in theabsence of osmotic pressure but resistance increased to greater than 800μg/ml in the presence of 300 mM NaCl. It was surprising that althoughthe P_(ompC1) allele was engineered from the chromosomal locus of E.coli, it appeared to function more efficiently in S. typhi. Theuninduced level of expression of GFPuv was the same for both DH5α andCVD 908-htrA (mean fluorescence intensity of 4.45 vs 5.37 respectively,Table 3). However, GFPuv synthesis increased 70% in DH5α afterinduction, but rose over 300% in CVD 908-htrA (mean fluorescenceintensity of 7.69 vs 23.4 respectively). This effect was not limited tothe P_(ompC1) allele but was equally remarkable when using P_(ompC3)(Table 3). These data do not agree with recent observations ofMartinez-Flores et al (1999) who reported that E. coli ompC-lacZ geneticfusions expressed constitutively within S. typhi, and that thisconstitutive level of expression was comparable to induced levels withinE. coli. Although we have identified a defined locus of point mutationsat the 3′-terminus of our E. coli P_(ompC) allele which could explainits osmotically controlled behavior within S. typhi CVD 908-htrA, suchmutations were not identified within P_(ompC3), which also responds toosmolarity within CVD 908-htrA. It should be noted, however, that thegenetic fusions studied by Martinez-Flores et al involved 1,150 bp ofthe E. coli 5′ ompC upstream control region, while the P_(ompC) allelesconstructed here involve only 459 bp of the 5′-proximal control regionof ompC. Regardless of this discrepancy, it is encouraging that thehighest levels of regulated heterologous gene expression are observedwithin the attenuated S. typhi live vector vaccine strain.

[0293] The contributions of several plasmid maintenance systems to thestability of plasmids within CVD 908-htrA, growing in the absence ofantibiotic selection, were then examined. No combination of maintenancefunctions could stabilize plasmids containing oriE1 origins ofreplication; in fact, these constructs were difficult to propagate evenin the presence of antibiotic. These observations cast doubt upon therationale for using higher copy number plasmids to optimize expressionof heterologous antigens within the cytoplasm of S. typhi-based livevectors, a strategy that, heretofore, has been followed by other groupsinvestigating Salmonella as live vectors (Covone et al. 1998).

[0294] Incorporation of plasmid maintenance systems into plasmidscarrying an ori15A origin of replication was more encouraging. When livevectors carrying such plasmids were passage without selection for 24 hrat 37° C., the effects of various combinations of maintenance functionsbecame apparent. In the absence of maintenance functions, the ori15Areplicon pGEN91 was lost from greater than 90% of the population,regardless of the level of induction of P_(ompC1) (see Table 6 and FIG.8). With incorporation of the hok-sok post-segregational killing locusin pGEN111, the percentage of bacteria expressing GFPuv tripled underall induction conditions, confirming the observations of others that thehok-sok locus enhances the stability of ori15A replicons (Gerdes et al.1985; Gerdes, 1988; Gerdes et al. 1997b). However, it was still notedthat regardless of induction conditions, greater than 50% of thebacterial population no longer fluoresced. Since it was confirmed thatat least a portion of this non-fluorescing population was still viableand lacked drug resistance, these data confirm previous reports (Gerdeset al. 1986; Wu and Wood, 1994; Pecota et al. 1997) that the presence ofa hok-sok post-segregational killing system is insufficient by itself toensure that plasmidless viable bacteria will not arise in a growingpopulation.

[0295] One possible mechanism that allows for escape from the influenceof hok-sok involves spontaneous point mutations arising within thelethal Hok open reading frame, which could conformationally inactivateHok and thereby allow plasmid loss to occur without lethality. Thispoint emphasizes the requirement of multiple mechanisms for enhancingthe stability of resident plasmids within growing bacteria; should onemaintenance function become inactivated, the probability of otherindependent functions simultaneously becoming inactivated becomesvanishingly small. Indeed, such redundancy in maintenance functions iswidespread within naturally occurring low copy number plasmids(Nordstrom and Austin, 1989). For example, the Escherichia coli sexfactor F contains one active partitioning function (sop) and two killingsystems (ccd and film) (Loh et al. 1988; Golub and Panzer, 1988; VanMelderen et al. 1994; Niki and Hiraga, 1997). Similarly, the drugresistance plasmid pR1 contains the active partitioning function parA,as well as the post-segregational killing system hok-sok; in addition,it carries yet another recently defined kis-kid killing system (Bravo etal. 1987; Bravo et al. 1988; Ruiz-Echevarria et al. 1995). Wedemonstrate in work reported here that insertion into multicopy ori15Areplicons of a more complete maintenance system, composed of both apost-segregational system and two partition functions, dramaticallyimproves the stability of these expression plasmids in the absence ofselection, regardless of induction conditions for heterologous antigenexpression. However, after passage without selection for 48 hrs,plasmids were eventually lost from the bacterial population, due toescape from the lethality of Hok. This problem has recently beenaddressed by Pecota et al (1997) who reported that incorporation of dualkilling systems significantly improved plasmid stability when comparedto the use of hok-sok alone; no partition functions were present inthese plasmids. Perhaps inclusion of the kis-kid killing system, to morefully represent the complement of pR1 stability functions, may berequired for optimal stability of higher copy expression plasmids withinS. typhi live vectors; since phd-doc PSK cassettes have recently beenconstructed, we are also examining the compatibility of this PSKfunction in our expression plasmids pGEN211, pGEN222 and pGEN206.

[0296] A comparison of strains carrying pGEN121 (an ori15A repliconcarrying hok-sok+par; ˜15 copies per chromosomal equivalent) with themuch lower copy number plasmid pGEN142 (an ori101 replicon carryinghok-sok+par; ˜5 copies per chromosomal equivalent) shows that underconditions of maximum induction of P_(ompC1) with 300 mM NaCl, 57% of apopulation of CVD 908-htrA(pGEN121), passaged for only 24 hr withoutselection, fluoresce with a mean fluorescence intensity of 105.3; for apopulation of CVD 908-htrA(pGEN142), passaged for 96 hr withoutselection under identical induction conditions, 94% of the bacteriaanalyzed by flow cytometry still maintain a mean fluorescence intensityof 47.7. Based on such results with GFPuv as a test antigen, it istempting to speculate that an optimum level of heterologous antigenpresented by an attenuated S. typhi-based live vector vaccine to thehuman immune system can be achieved by decreasing the copy number ofresident expression plasmids to perhaps 5 copies per chromosomalequivalent.

[0297] The efficiency of eliciting an immune response directed against aheterologous antigen will depend in part upon the ability of the livevector to present such antigens to the immune system. The ability of alive vector to present antigens will in turn depend upon the stabilityof multicopy expression plasmids that encode the heterologous antigens.Our results demonstrate that inclusion of a plasmid maintenance systemwithin multicopy expression plasmids, without further geneticmanipulation of the live vector, enhances the stability of suchexpression plasmids. However, the presence of multicopy plasmids mayalso influence the metabolic fitness of the live vector. This isrelevant because some foreign antigens of interest exert a deleteriouseffect on the live vector.

[0298] While we do not intend to be bound to this theory, we concludethat a significant metabolic burden is placed upon CVD 908-htrA carryinga multicopy expression plasmid; as copy number and/or level of geneexpression increases, metabolic burden increases. Studies with E. colihave clearly established that plasmid-bearing bacteria grow slower thanplasmidless bacteria (Boe et al. 1987; McDermott et al. 1993; Wu andWood, 1994; Pecota et al. 1997; Summers, 1998). It has also beendemonstrated that as copy number increases, the growth rate of suchstrains decreases; similarly, as induction of heterologous genesincreases, growth rate decreases further (Wu and Wood, 1994; Pecota etal. 1997). Clearly, spontaneous plasmid loss would remove any metabolicburden and allow plasmidless bacteria to quickly outgrow the populationof plasmid-bearing bacteria. In elegant studies, Wu and Wood (Wu andWood, 1994) showed that plasmid-bearing E. coli strains maintainedplasmids under conditions where cloned gene expression was low for 100hr when passaged in the absence of selection; in contrast, under maximuminduction conditions, complete plasmid loss occurred within 10 hr.Interestingly, when the hok-sok locus was inserted into these expressionplasmids, the plasmids were maintained for 300 hr. under uninducedconditions and 30 hr. under inducing conditions. Such a shift in antigenexpression within a population of live vector bacteria would be expectedto reduce the efficiency of stimulating any immune response specific tothe foreign antigen. Our analysis leads us to conclude that the goal foran effective multivalent S. typhi-based live vector vaccine is tooptimize viability using stabilized lower copy number expressionvectors, capable of expressing high levels of heterologous antigen inresponse to an environmental signal likely to be encountered in vivoafter the vaccine organisms have reached an appropriate ecologicalniche. We are currently testing this strategy using the murineintranasal model to examine the immunogenicity of fragment C of tetanustoxin expressed within CVD 908-htrA from our expression vectors pGEN211(oriE1), pGEN222 (ori15A), and pGEN206 (ori101), all of which carryidentical plasmid maintenance systems and differ only in copy number.The work presented herein enables the development of single dose, oralS. typhi-based live vector vaccines capable of inducing protectiveimmune responses against multiple unrelated human pathogens.

7. REFERENCES

[0299] The disclosures of the following references are incorporatedherein in their entirety:

[0300] Acheson, D. W. K. 1998. Nomenclature of enterotoxins. Lancet351:1003.

[0301] Acheson, D. W. K., M. M. Levine, J. B. Kaper, and G. T. Keusch.1996. Protective immunity to Shiga-like toxin I following oralimmunization with Shiga-like toxin I B-subunit-producing Vibrio choleraeCVD 103-HgR. Infection and Immunity 64:355.

[0302] Austin, S. J. 1988. Plasmid partition. Plasmid 20:1.

[0303] Austin, S., S. Friedman, and D. Ludtke. 1986. Partition functionsof unit-copy plasmids can stabilize the maintenance of plasmid pBR322 atlow copy number. J Bacteriol 168: 1010-1013.

[0304] Barry, E. M., O. G. Gomez-Duarte, S. Chaffield, R. Rappuoli, M.Pizza, G. Losonsky, J. E. Galen, and M. M. Levine. 1996. Expression andimmunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment Cfusions in Salmonella typhi vaccine strain CVD 908. Infection andImmunity 64:41724181

[0305] Barth, P. T., H. Richards, and N. Datta. 1978. Copy numbers ofcoexisting plasmids in Escherichia coli K-12. J Bacteriol 135: 760-765.

[0306] Bast, D. J., J. L. Brunton, M. A. Karmali, and S. E. Richardson.1997. Toxicity and immunogenicity of a verotoxin 1 mutant with reducedglobotriaosylceramide receptor binding in rabbits. Infection andImmunity 65:2019.

[0307] Baumler, A. J., J. G. Kusters, I. Stojiljkovic, and F. Heffron.1994. Salmonella typhimurium loci involved in survival withinmacrophages. Infection and Immunity 62:1623.

[0308] Beaucage, S. L., C. A. Miller, and S. N. Cohen. 1991.Gyrase-dependent stabilization of pSC101 plasmid inheritance bytranscriptionally active promoters. EMBO J 10: 2583-2588.

[0309] Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V.Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F.Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J.Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence ofEscherichia coli K-12. Science 277:1453.

[0310] Blomfield, I. C., V. Vaughn, R. F. Rest, and B. I. Eisenstein.1991. Allelic exchange in Escherichia coli using the Bacillus subtilissacB gene and a temperature-sensitive pSC101 replicon. MolecularMicrobiology 5:1447-1457.

[0311] Boe, L. and K. V. Rasmussen. 1996. Suggestions as to quantitativemeasurements of plasmid loss. Plasmid 36:153.

[0312] Boe, L., K. Gerdes, and S. Molin. 1987. Effects of genes exertinggrowth inhibition and plasmid stability on plasmid maintenance. Journalof Bacteriology 169:4646-4650.

[0313] Bokman, S. H. and W. W. Ward. 1981. Renaturation of Aequoreagreen-fluorescent protein. Biochemical and Biophysical ResearchCommunications 101:1 372.

[0314] Bosworth, B. T., J. E. Samuel, H. W. Moon, A. D. O'Brien, V. M.Gordon, and S. C. Whipp. 1996. Vaccination with genetically modifiedShiga-like toxin lie prevents edema disease in swine. Infection andImmunity 64:55.

[0315] Bouvier, J., C. Richaud, W. Higgins, O. Bogler, and P. Stragier.1992. Cloning, characterization, and expression of the dapE gene ofEscherichia coli. Journal of Bacteriology 174:5265.

[0316] Boyd, B. and C. A. Lingwood. 1989. Verotoxin receptor glycolipidin human renal tissue. Nephron 51:207.

[0317] Bravo, A., G. de Torrontegui, and R. Diaz. 1987. Identificationof components of a new stability system of plasmid R1, ParD, that isclose to the origin of replication of this plasmid. Mol Gen Genet 210:101-110.

[0318] Bravo, A., S. Ortega, G. de Torrontegui, and R. Diaz. 1988.Killing of Escherichia coli cells modulated by components of thestability system parD of plasmid R1. Mol Gen Genet 215: 146-151.

[0319] Brosius, J. 1989. Superpolylinkers in cloning and expressionvectors. DNA 8: 759-777.

[0320] Butterton, J. R., E. T. Ryan, D. W. Acheson, and S. B.Calderwood. 1997. Coexpression of the B subunit of Shiga toxin 1 andEaeA from enterohemorrhagic Escherichia coli in Vibrio cholerae vaccinestrains. Infection and Immunity 65:2127-2135

[0321] Cabello, F., K. Timmis, and S. N. Cohen. 1976. Replicationcontrol in a composite plasmid constructed by in vitro linkage of twodistinct replicons. Nature 259: 285-290.

[0322] Calderwood, S. B., D. W. K. Acheson, G. T. Keusch, T. J. Barrett,P. M. Griffin, N. A. Strockbine, B. Swaminathan, J. B. Kaper, M. M.Levine, B. S. Kaplan, H. Karch, A. D. O'Brien, T. G. Obrig, Y. Takeda,P. I. Tarr, and I. K. Wachsmuth. 1996. Proposed new nomenclature for SLT(VT) family. ASM News 62:118.

[0323] Calderwood, S. B., F. Auclair, A. Donohue-Rolfe, G. T. Keusch,and J. J. Mekalanos. 1987. Nucleotide sequence of the Shiga-like toxingenes of Escherichia coli. Proceedings of the National Academy ofSciences USA 84:4364.

[0324] Carlini, L. E., R. D. Porter, U. Curth, and C. Urbanke. 1993.Viability and preliminary in vivo characterization of site-specificmutants of Escherichia coli single-stranded DNA-binding protein.Molecular Microbiology 10:1067.

[0325] Carter, P. B. and F. M. Collins. 1974. Growth of typhoid andparatyphoid bacilli in intravenously infected mice. Infection andImmunity 10:816.

[0326] Cerin, H. and J. Hackett. 1989. Molecular cloning and analysis ofthe incompatibility and partition functions of the virulence plasmid ofSalmonella typhimurium. Microbial Pathogenesis 7:85.

[0327] Cerin, H. and J. Hackett. 1993. The parVP region of theSalmonella typhimurium virulence plasmid pSLT contains four locirequired for incompatibility and partition. Plasmiid 30:30.

[0328] Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher.1994. Green fluorescent protein as a marker for gene expression. Science263:802.

[0329] Chambers, S. P., S. E. Prior, D. A. Barstow, and N. P. Minton.1988. The pMTLnic cloning vectors. I. Improved pUC polylinker regions tofacilitate the use of sonicated DNA for nucleotide sequencing. Gene68:139.

[0330] Chang, A. C. Y., and S. N. Cohen. 1978. Construction andcharacterization of amplifiable multicopy DNA cloning vehicles derivedfrom the P15A cryptic miniplasmid. J Bacteriol 134: 1141-1156.

[0331] Chase, J. W. and K. R. Williams. 1986. Single-stranded DNAbinding proteins required for DNA replication. Annual Reviews inBiochemistry 55:103.

[0332] Chase, J. W., J. B. Murphy, R. F. Whittier, E. Lorensen, and J.J. Sninsky. 1983. Amplification of ssb-1 mutant single-strandedDNA-binding protein in Escherichia coli. Journal of Molecular Biology163,164:193.

[0333] Chatfield, S., K. Strahan, D. Pickard, I. G. Charles, C. E.Hormaeche, and G. Dougan. 1992. Evaluation of Salmonella typhimuriumstrains harbouring defined mutations in htrA and aroA in the murinesalmonellosis model. Microbial Pathogenesis 12:145.

[0334] Clark, C., D. Bast, A. M. Sharp, P. M. St.Hilaire, R. Agha, P. E.Stein, E. J. Toone, R. J. Read, and J. L. Brunton. 1996. Phenylalanine30 plays an important role in receptor binding of verotoxin-1. MolecularMicrobiology 19:891.

[0335] Conradi, H. 1903. Ueber losliche,durch aseptische autolyseerhaltene giftstoffe von ruhr-und Typhusbazillen. Dtsch. Med.Wochenschr. 29:26.

[0336] Covarrubias, L., L. Cervantes, A. Covarrubias, X. Soberon, I.Vichido, A. Blanco, Y. M. Kupersztoch -Portnoy, and F. Bolivar. 1981.Construction and characterization of new cloning vehicles. V.Mobilization and coding properties of pBR322 and several deletionderivatives including pBR327 and pBR328. Gene 13: 25-35.

[0337] Covone, M. G., M. Brocchi, E. Palla, W. D. da Silveira, R.Rappuoli, and C. L. Galeotti. 1998. Levels of expression andimmunogenicity of attenuated Salmonella enterica serovar typhimuriumstrains expressing Escherichia coli mutant heat-labile enterotoxin.Infection and Immunity 66:224-231

[0338] Crameri, A., E. A. Whitehorn, E. Tate, and W. P. Stemmer. 1996.Improved green fluorescent protein by molecular evolution using DNAshuffling. Nat Biotechnol 14: 315-319.

[0339] Dam, M. and K. Gerdes. 1994. Partitioning of plasmid R1: tendirect repeats flanking the parA promoter constitute a centromere-likepartition site parC, that expresses incompatibility. Journal ofMolecular Biology 236:1289-1298.

[0340] Dopf, J. and T. M. Horiagon. 1996. Deletion mapping of theAequorea victoria green fluorescent protein. Gene 173:39.

[0341] Downes, F. P., T. J. Barrett, J. H. Green, C. H. Aloisio, J. S.Spika, N. A. Strockbine, and I. K. Wachsmuth. 1988. Affinitypurification and characterization of Shiga-like toxin II and productionof toxin-specific monoclonal antibodies. Infection and Immunity 56:1926.

[0342] Egger, L. A., H. Park, and M. Inouye. 1997. Signal transductionvia the histidyl-aspartyl phosphorelay. Genes to Cells 2:167.

[0343] Endo, Y., K. Tsurugi, T. Yutsudo, Y. Takeda, T. Ogasawara, and K.Igarashi. 1988. Site of action of a Vero toxin (VT2) from Escherichiacoli O157:H7 and of Shiga toxin on eukaryotic ribosomes: RNAN-glycosidase activity of the toxins. European Journal of Biochemistry171:45.

[0344] Forrest, B. D., J. T. Labrooy, S. R. Attridge, G. Boehm, L.Beyer, R. Morona, D. J. C. Shearman, and D. Rowley. 1989. Immunogenicityof a candidate live oral typhoid/cholera hybrid vaccine in humans. J.Infect. Dis. 159: 145.

[0345] Fraser, M. E., M. M. Chernaia, Y. V. Koziov, and M. N. G. James.1994. Crystal structure of the holotoxin from Shigella dysenteriae at2.5 A resolution. Nature Structural Biology 1:59.

[0346] Galan, J. E., K. Nakayama, and R. Curtiss III. 1990. Cloning andcharacterization of the asd gene of Salmonella typhimurium: use instable maintenance of recombinant plasmids in Salmonella vaccinestrains. Gene 94:29-35.

[0347] Galen, J. E. and M. M. Levine. 1995. Improved suicide vectors forchromosomal mutagenesis in Salmonella typhi. Abstracts of the AnnualMeeting of the American Society of Microbiology H192:(Abstract)

[0348] Galen, J. E. and M. M. Levine. 1996. Further refinements ofsuicide vector-mediated chromosomal mutagenesis in Salmonella typhi.Abstracts of the Annual Meeting of the American Society of MicrobiologyH260: (Abstract)

[0349] Galen, J. E., O. G. Gomez-Duarte, G. Losonsky, J. L. Halpern, C.S. Lauderbaugh, S. Kaintuck, M. K. Reymann, and M. M. Levine. 1997. Amurine model of intranasal immunization to assess the immunogenicity ofattenuated Salmonella typhi live vector vaccines in stimulating serumantibody responses to expressed foreign antigens. Vaccine 15:700-708.

[0350] Galen, J. E., E. R. Vimr, L. Lawrisuk, and J. B. Kaper. 1990.Cloning, sequencing, and expression of the gene, nanH, for Vibriocholerae neuraminidase. In Advances in research on cholera and relateddiarrheas (Edited by Sack R. B. and Zinnake Y. Tokyo: KTK ScientificPublishers. pp. 143-153.

[0351] Gay, P., D. Le Coq, M. Steinmetz, E. Ferrari, and J. A. Hoch.1983. Cloning structural gene sacB, which codes for exoenzymelevansucrase of Bacillus subtilis: expression of the gene in Escherichiacoli. Journal of Bacteriology 153:1424.

[0352] Gerdes, K. 1988. The parB (hok-sok) locus of plasmid R1: ageneral purpose plasmid stabilization system. Bio/Technology 6:1402-1405.

[0353] Gerdes, K. and S. Molin. 1986. Partitioning of plasmid R1:structural and functional analysis of the parA locus. Journal ofMolecular Biology 190:269.

[0354] Gerdes, K., A. P. Gultyaev, T. Franch, K. Pedersen, and N. D.Mikkelsen. 1997. Antisense RNA-regulated programmed cell death. AnnualReviews in Genetics 31:1-31.

[0355] Gerdes, K., J. S. Jacobsen, and T. Franch. 1997b. Plasmidstabilization by post-segregational killing. Genet Eng (NY) 19: 49-61.

[0356] Gerdes, K., J. E. Larsen, and S. Molin. 1985. Stable inheritanceof plasmid R1 requires two different loci. J Bacteriol 161: 292-298.

[0357] Gerdes, K., P. B. Rasmussen, and S. Molin. 1986. Unique type ofplasmid maintenance function: postsegregational killing of plasmid-freecells. Proc Natl Acad Sci USA 83: 3116-3120.

[0358] Gerichter, C. B. 1960. The dissemination of Salmonella typhi, S.paratyphi A, and S. paratyphi B through the organs of the white mouse byoral infection. Journal of Hygiene, Cambridge 58:307.

[0359] Gerichter, C. B. and D. L. Boros. 1962. Dynamics of infection ofthe blood stream and internal organs of white mice with Salmonella typhiby intraperitoneal injection. Journal of Hygiene, Cambridge 60:311.

[0360] Golub, E. I., and H. A. Panzer. 1988. The F factor of Escherichiacoli carries a locus of stable plasmid inheritance stm, similar to theparB locus of plasmid R1. Mol Gen Genet 214: 353-357.

[0361] Gomez-Duarte, O. G., J. E. Galen, S. N. Chatfield, R. Rappuoli,L. Eidels, and M. M. Levine. 1995.

[0362] Expression of fragment C of tetanus toxin fused to acarboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD908 vaccine strain. Vaccine 13:1596.

[0363] Gonzalez, C., D. M. Hone, F. Noriega, C. O. Tacket, J. R. Davis,G. Losonsky, J. P. Nataro, S. Hoffman, A. Malik, E. Nardin, M. Sztein,D. G. Heppner, T. R. Fouts, A. Isibasi, and M. M. Levine. 1994.Salmonella typhi vaccine strain CVD 908 expressing the circumsporozoiteprotein of Plasmodium falciparum: strain construction and safety andimmunogenicity in humans. Journal of Infectious Diseases 169:927-931.

[0364] Gordon, V. M., S. C. Whipp, H. W. Moon, A. D. O'Brien, and J. E.Samuel. 1992. An enzymatic mutant of Shiga-like toxin II variant is avaccine candidate for edema disease of swine. Infection and Immunity60:485.

[0365] Gottesman, S., W. P. Clark, V. de Crecy-Lagard, and M. R.Maurizi. 1993. ClpX, an alternative subunit for the ATP-dependent Clpprotease of Escherichia coli. Journal of Biological Chemistry 268:22618.

[0366] Green, J. M., B. P. Nichols, and R. G. Matthews. 1996. Folatebiosynthesis, reduction, and polyglutamylation. In Escherichia coli andSalmonella: Cellular and molecular biology. 2nd ed. F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington, D.C. p. 665.

[0367] Griffin, P. M. 1995. Escherichia coli O157:H7 and otherenterohemorrhagic Escherichia coli. In Infections of thegastrointestinal tract. M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B.Greenberg and R. L. Guerrant, eds. Raven Press, Ltd, New York, p. 739.

[0368] Gyles, C. L. 1992. Escherichia coli cytotoxins and enterotoxins.Canadian Journal of Microbiology 38:734.

[0369] Heim, R., D. C. Prasher, and R. Y. Tsien. 1994. Wavelengthmutations and posttranscriptional autoxidation of green fluorescentprotein. Proceedings of the National Academy of Sciences USA 91:12501.

[0370] Hiszczynska-Sawicka, E., and J. Kur. 1997. Effect of Escherichiacoli IHF mutations on plasmid p15A copy number. Plasmid 38: 174-179.

[0371] Hoiseth, S. K. and B. A. Stocker. 1981. Aromatic-dependentSalmonella typhimurium are non-virulent and effective as live vaccines.Nature 291:238.

[0372] Hone, D. M., A. M. Harris, S. Chatfield, G. Dougan, and M. M.Levine. 1991. Construction of genetically defined double aro mutants ofSalmonella typhi. Vaccine 9: 810-816.

[0373] Hovde, C. J., S. B. Calderwood, J. J. Mekalanos, and R. J.Collier. 1988. Evidence that glutamic acid 167 is an active-site residueof Shiga-like toxin I. Proceedings of the National Academy of SciencesUSA 85:2568.

[0374] Jackson, M. P., E. A. Wadolkowski, D. L. Weinstein, R. K. Holmes,and A. D. O'Brien. 1990. Functional analysis of the Shiga toxin andShiga-like toxin type II variant binding subunits by using site-directedmutagenesis. Journal of Bacteriology 172:653.

[0375] Jackson, M. P., R. J. Neill, A. D. O'Brien, R. K. Holmes, and J.W. Newland. 1987. Nucleotide sequence analysis and comparison of thestructural genes for Shiga-like toxin I and Shiga-like toxin II encodedby bacteriophages from Escherichia coli. FEMS Microbiology Letters44:109.

[0376] Jackson, M. P., R. L. Deresiewicz, and S. B. Calderwood. 1990.Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymaticsubunits. Journal of Bacteriology 172:3346.

[0377] Jarvis, K. G. and J. B. Kaper. 1996. Secretion of extracellularproteins by enterohemorrhagic Escherichia coli via a putative type IIIsecretion system. Infection and Immunity 64:4826.

[0378] Jarvis, K. G., J. A. Giron, A. E. Jerse, T. K. McDaniel, M. S.Donnenberg, and J. B. Kaper. 1995. Enteropathogenic Escherichia colicontains a putative type III secretion system necessary for the exportof proteins involved in attaching and effacing lesion formation.Proceedings of the National Academy of Sciences USA 92:7996.

[0379] Jensen, R. B. and K. Gerdes. 1995. Programmed cell death inbacteria: proteic plasmid stabilization systems. Molecular Microbiology17:205.

[0380] Jensen, R. B. and K. Gerdes. 1997. Partitioning of plasmid R1.The ParM protein exhibits ATPase activity and interacts with thecentromere-like ParR-parC complex. Journal of Molecular Biology269:505-513.

[0381] Karem, K. L., S. Chatfield, N. Kuklin, and B. T. Rouse. 1995.Differential induction of carrier antigen-specific immunity bySalmonella typhimurium live-vaccine strains after single mucosal orintravenous immunization of BALB/c mice. Infection and Immunity63:4557-4563.

[0382] Karmali, M. A. 1989. Infection by verocytotoxin-producingEscherichia coli. Clinical Microbiological Reviews 2:15.

[0383] Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, and B. T.Steele. 1983. Escherichia coli cytotoxin, haemolytic-uraemic syndrome,and haemorrhagic colitis. Lancet ii: 1299.

[0384] Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus,and H. Lior. 1985. The association between idiopathic hemolytic uremicsyndrome and infection by verotoxin-producing Escherichia coli. Journalof Infectious Diseases 151:775.

[0385] Karpman, D., H. Connell, M. Svensson, F. Scheutz, P. Alm, and C.Svanborg. 1997. The role of lipopolysaccharide and Shiga-like toxin in amouse model of Escherichia coli O157:H7 infection. Journal of InfectiousDiseases 175:611.

[0386] Keusch, G. T., G. F. Grady, L. J. Mata, and J. McIver. 1972.Pathogenesis of shigella diarrhea. 1. Enterotoxin production by Shigelladysenteriae 1. Journal of Clinical Investigation 51:1212.

[0387] Killeen, K. P., V. Escuyer, J. J. Mekalanos, and R. J. Collier.1992. Reversion of recombinant toxoids: mutations in diphtheria toxinthat partially compensate for active-site deletions. Proceeding of theNational Academy of Sciences USA 89:6207.

[0388] Kim, J. Y., H. A. Kang, and D. D. Ryu. 1993. Effects of the parlocus on the growth rate and structural stability of recombinant cells.Biotechnology Progress 9:548.

[0389] Konowalchuk, J. , J. I. Speirs, and S. Stavric. 1977. Veroresponse to a cytotoxin of Escherichia coli. Infection and Immunity18:775.

[0390] Langermann, S., S. Palaszynski, A. Sadziene, C. K. Stover, and S.Koenig. 1994. Systemic and mucosal immunity induced by BCG vectorexpressing outer-surface protein A of Borrelia burgdorferi. Nature 372:552-555.

[0391] Lee, S. F., R. J. March, S. A. Halpern, G. Faulkner, and L. Gao.1999. Surface expression of a protective recombinant pertussis toxin S1subunit fragment in Streptococcus gordonii. Infect Immun 67: 1511-1516.

[0392] Lehnherr, H. and M. B. Yarmolinsky. 1995. Addiction protein Phdof plasmid prophage P1 is a substrate of the ClpXP serine protease ofEscherichia coli. Proceedings of the National Academy of Sciences USA92:3274.

[0393] Lehnherr, H., E. Maguin, S. Jafri, and M. B. Yarmolinsky. 1993.Plasmid addiction genes of bacteriophage P1: doc, which causes celldeath on curing of prophage, and phd, which prevents host death whenprophage is retained. Journal of Molecular Biology 233:414.

[0394] Levine, M. M., J. E. Galen, E. M. Barry, F. Noriega, S.Chatfield, M. Sztein, G. Dougan, and C. O. Tacket. 1996. AttenuatedSalmonella as live oral vaccines against typhoid fever and as livevectors. Journal of Biotechnology 44:193.

[0395] Lindgren, S. W., J. E. Samuel, C. K. Schmitt, and A. D. O'Brien.1994. The specific activities of Shiga-like toxin type II (SLT-II) andSLT-II-related toxins of enterohemorrhagic Escherichia coli differ whenmeasured by Vero cell cytotoxicity but not by mouse lethality. Infectionand Immunity 62:623.

[0396] Lloyd, R. G. and K. B. Low. 1996. Homologous recombination. InEscherichia coli and Salmonella: Cellular and molecular biology. 2nd ed.F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B.Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.Umbarger, eds. ASM Press, Washington, D.C. p. 2236.

[0397] Loh, S. M., D. S. Cram, and R. A. Skurray. 1988. Nucleotidesequence and transcriptional analysis of a third function (Flm) involvedin F plasmid maintenance. Gene 66: 259-268.

[0398] Lohman, T. M. and M. E. Ferrari. 1994. Escherichia colisingle-stranded DNA-binding protein: multiple DNA-binding modes andcooperativities. Annual Reviews in Biochemistry 63:527.

[0399] Louise, C. B. and T. G. Obrig. 1995. Specific interaction ofEscherichia coli O157:H7-derived Shiga-like toxin II with human renalendothelial cells. Journal of Infectious Diseases 172:1397.

[0400] Love, C. A., P. E. Lilley, and N. E. Dixon. 1996. Stablehigh-copy-number bacteriophage lambda promoter vectors foroverproduction of proteins in Escherichia coli. Gene 176:49.

[0401] Lynch, A. S. and E. C. C. Lin. 1996. Responses to molecularoxygen. In Escherichia coli and Salmonella: Cellular and molecularbiology. 2nd ed. F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C.C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.Schaechter and H. E. Umbarger, eds. ASM Press, Washington, D.C. p. 1526.

[0402] Magnuson, R., H. Lehnherr, G. Mukhopadhyay, and M. B.Yarmolinsky. 1996. Autoregulation of the plasmid addiction operon ofbacteriophage P1. Journal of Biological Chemistry 271:18705.

[0403] Makoff, A. J., and A. E. Smallwood. 1988. Heterologous expressionin Escherichia coli: effects of alterations in the sequence 5′ to theinitiation codon. Biochem Soc Trans 16: 4849.

[0404] Mangeney, M., C. A. Lingwood, S. Taga, B. Caillou, T. Tursz, andJ. Wiels. 1993. Apoptosis induced in Burkitt's lymphoma cells viaGb₃/CD77, a glycolipid antigen. Cancer Research 53:5314.

[0405] Marshall, J., R. Molloy, G. W. J. Moss, J. R. Howe, and T. E.Hughes. 1995. The jellyfish green fluorescent protein: a new tool forstudying ion channel expression and function. Neuron 14:211.

[0406] Martinez-Flores, I., R. Cano, V. H. Bustamante, E. Calva, and J.L. Puente. 1999. The ompB operon partially determines differentialexpression of OmpC in Salmonella typhi and Escherichia coli. J Bacteriol181: 556-562.

[0407] Matthews, R. G. 1996. One-carbon metabolism. In Escherichia coliand Salmonella: Cellular and molecular biology. 2nd ed. F. C. Neidhardt,R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik,W. S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington, D.C. p. 600.

[0408] Maurizi, M. R., W. P. Clark, Y. Katayama, S. Rudikoff, J.Pumphrey, B. Bowers, and S. Gottesman. 1990. Sequence and structure ofClp P, the proteolytic component of the ATP-dependent Clp protease ofEscherichia coli. Journal of Biological Chemistry 265:12536.

[0409] McClelland, M. and R. Wilson. 1998. Sample sequencing of theSalmonella typhi genome: comparison to the E. coli K-12 genome.Infection and Immunity

[0410] McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper.1995. A genetic locus of enterocyte effacement conserved among diverseenterobacterial pathogens. Proceedings of the National Academy ofSciences USA 92:1664.

[0411] McDermott, P. J., P. Gowland, and P. C. Gowland. 1993. Adaptationof Escherichia coli growth rates to the presence of pBR322. Lett ApplMicrobiol 17: 139-143.

[0412] Meacock, P. A., and S. N. Cohen. 1980. Partitioning of bacterialplasmids during cell division: a cis-acting locus that accomplishesstable plasmid inheritance. Cell 20: 529-542.

[0413] Medaglini, D., G. Pozzi, T. P. King, and V. A. Fischetti. 1995.Mucosal and systemic immune responses to a recombinant protein expressedon the surface of the oral commensal bacterium Streptococcus gordoniiafter oral colonization. Proc Natl Acad Sci USA 92: 6868-6872.

[0414] Melton-Celsa, A. R. and A. D. O'Brien. 1998. The structure,biology, and relative toxicity for cells and animals of Shiga toxinfamily members. In Escherichia coli O157:H7 and other Shigatoxin-producing E. coli strains. J. B. Kaper and A. D. O'Brien, eds. ASMPress, Washington, D.C. In press.

[0415] Mikkelsen, N. D. and K. Gerdes. 1997. Sok antisense RNA fromplasmid R1 is functionally inactivated by RNaseE and polyadenylated bypoly(A) polymerase I. Molecular Microbiology 26:311.

[0416] Miller, C. A., S. L. Beaucage, and S. N. Cohen. 1990. Role of DNAsuperhelicity in partitioning of the pSC101 plasmid. Cell 62: 127-133.

[0417] Moxley, R. A. and D. H. Francis. 1998. Overview of Animal Models.In Escherichia coli O157:H7 and other Shiga toxin-producing E. colistrains. J. B. Kaper and A. D. O'Brien, eds. ASM Press, Washington, D.C.In press.

[0418] Muhldorfer, I., J. Hacker, G. T. Keusch, D. W. Acheson, H.Tschape, A. V. Kane, A. Ritter, T. Olschlager, and A. Donohue-Rolfe.1996. Regulation of the Shiga-like toxin 11 operon in Escherichia coli.Infection and Immunity 64:495.

[0419] Nakayama, K., S. M. Kelley, and R. Curtiss III. 1988.Construction of an Asd⁺ expression-cloning vector: stable maintenanceand high level expression of cloned genes in a Salmonella vaccinestrain. Bio/Technology 6: 693-697.

[0420] Nakayama, K., S. M. Kelley, and R. Curtiss III. 1988.Construction of an Asd⁺ expression-cloning vector: stable maintenanceand high level expression of cloned genes in a Salmonella vaccinestrain. Bio/Technology 6:693.

[0421] Nelson, S., S. E. Richardson, C. A. Lingwood, M. Petric, and M.A. Karmali. 1994. Biological activity of verocytotoxin (VT)2c andVT1/VT2c chimeras in the rabbit model. In Recent advances inverocytotoxin-producing Escherichia Coli infections. M. A. Karmali andA. G. Goglio, eds. Elsevier Science, New York, p. 245.

[0422] Niki, H., and S. Hiraga. 1997. Subcellular distribution ofactively partitioning F plasmid during the cell division cycle of E.coli. Cell 90: 951-957.

[0423] Nordstrom, K. and S. J. Austin. 1989. Mechanisms that contributeto the stable segregation of plasmids. Annual Reviews in Genetics 23:37.

[0424] Noriega, F. R., G. Losonsky, J. Y. Wang, S. B. Formal, and M. M.Levine. 1996. Further characterization of ΔaroA ΔvirG Shigella flexneri2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vectorvaccine for delivering antigens of enterotoxigenic Escherichia coli.Infect Immun 64: 23-27.

[0425] Norioka, S., G. Ramakrishnan, K. Ikenaka, and M. Inouye. 1986.Interaction of a transcriptional activator,OmpR, with reciprocallyosmoregulated genes, ompF and ompC, of Escherichia coli. Journal ofBiological Chemistry 261:17113-17119

[0426] Nyholm, P., G. Magnusson, Z. Zheng, R. Norel, B. Binnington-Boyd,and C. A. Lingwood. 1996. Two distinct binding sites for globotriaosylceramide on verotoxins: identification by molecular modelling andconfirmation using deoxy analogues and a new glycolipid receptor for allverotoxins. Chemistry and Biology 3:263.

[0427] Nyholm, P., J. L. Brunton, and C. A. Lingwood. 1995. Modelling ofthe interaction of verotoxin-1 (VT1) with its glycolipid receptor,globotriaosylceramide (Gb₃). International Journal of BiologicalMacromolecules 17:199.

[0428] O'Brien, A. D. 1982. Innate resistance of mice to Salmonellatyphi infection. Infection and Immunity 38:948.

[0429] O'Brien, A. D., V. L. Tesh, A. Donohue-Rolfe, M. P. Jackson, S.Olsnes, K. Sandvig, A. A. Lindberg, and G. T. Keusch. 1992. Shiga toxin:biochemistry,genetics,mode of action, and role in pathogenesis. CurrentTopics in Microbiology and Immunology 180:65.

[0430] Olitsky, P. K. and I. J. Kligler. 1920. Toxins and antitoxins ofBacillus dysenteriae Shiga. Journal of Experimental Medicine 31:19.

[0431] Orosz, A., I. Boros, and P. Venetianer. 1991. Analysis of thecomplex transcription termination region of the Escherichia coli rrnBgene. European Journal of Biochemistry 201:653.

[0432] Oxer, M. D., C. M. Bentley, J. G. Doyle, T. C. Peakman, I. G.Charles, and A. J. Makoff. 1991. High level heterologous expression inE. coli using the anaerobically-activated nirB promoter. Nucleic AcidsResearch 19:2889-2892.

[0433] Pallen, M. J. and B. W. Wren. 1997. The HtrA family of serineproteases. Molecular Microbiology 26:209.

[0434] Pecota, D. C., C. S. Kim, K. Wu, K. Gerdes, and T. K. Wood. 1997.Combining the hokisok, parDE, and pnd postsegregational killer loci toenhance plasmid stability. Applied and Environmental Microbiology63:1917-1924.

[0435] Perera, L. P., J. E. Samuel, R. K. Holmes, and A. D. O'Brien.1991. Mapping the minimal contiguous gene segment that encodesfunctionally active Shiga-like toxin II. Infection and Immunity 59:829.

[0436] Perera, L. P., J. E. Samuel, R. K. Holmes, and A. D. O'Brien.1991. Identification of three amino acid residues in the B subunit ofShiga toxin and Shiga-like toxin type II that are essential forholotoxin activity. Journal of Bacteriology 173:1151.

[0437] Pittard, A. J. 1996. Biosynthesis of the aromatic amino acids. InEscherichia coli and Salmonella: Cellular and molecular biology. 2nd ed.F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B.Low, B.

[0438] Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.Umbarger, eds. ASM Press, Washington, D.C. p. 458.

[0439] Polisky, B. 1986. Replication control of the ColE1-type plasmids.In Maximizing gene expression. W. S. Reznikoff and L. Gold, eds.Butterworths, Boston, p. 143.

[0440] Porter, R. D., S. Black, S. Pannuri, and A. Carlson. 1990. Use ofthe Escherichia coli ssb gene to prevent bioreactor takeover byplasmidless cells. Bio/Technology 8:47.

[0441] Pouwels, P. H., R. J. Leer, M. Shaw, M. J. Heijne denBak-Glashouwer, F. D. Tielen, E., Smit, B. Martinez, J. Jore, and P. L.Conway. 1998. Lactic acid bacteria as antigen delivery vehicles for oralimmunization purposes. Int J Food Microbiol 41: 155-167.

[0442] Pratt, L. A., W. Hsing, K. E. Gibson, and T. J. Silhavy. 1996.From acids to osmZ: mutiple factors influence synthesis of the OmpF andOmpC porins in Escherichia coli. Molecular Microbiology 20:911.

[0443] Puente, J. L., V. Alvarez-Scherer, G. Gosset, and E. Calva. 1989.Comparative analysis of the Salmonella typhi and Escherichia coli ompCgenes. Gene 83:197.

[0444] Richardson, S. E., T. A. Rotman, V. Jay, C. R. Smith, L. E.Becker, M. Petric, N. F. Olivieri, and M. A. Karmali. 1992. Experimentalverocytotoxemia in rabbits. Infection and Immunity 60:4154.

[0445] Ringquist, S., S. Shinedling, D. Barrick, L. Green, J. Binkley,G. D. Stormo, and L. Gold. 1992. Translation initiation in Escherichiacoli: sequences within the ribosome-binding site. Molecular Microbiology6:1219.

[0446] Roberts, M., S. Chatfield, and G. Dougan. 1994. Salmonella ascarriers of heterologous antigens. In Novel delivery systems for oralvaccines. D. T. O'Hagan, ed. CRC Press, Ann Arbor, p. 27-58.

[0447] Ruiz-Echevarria, M. J., G. Gimenez-Gallego, R. Sabariegos-Jareno,and R. Diaz-Orejas. 1995. Kid, a small protein of the parD stabilitysystem of plasmid R1, is an inhibitor of DNA replication acting at theinitiation of DNA synthesis. J Mol Biol 247: 568-577.

[0448] Rupp, W. D. 1996. DNA repair mechanisms. In Escherichia coli andSalmonella: Cellular and molecular biology. 2nd ed. F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington, D.C. p. 2277.

[0449] Ryan, E. T., J. R. Bufterton, R. N. Smith, P. A. Carroll, T. I.Crean, and S. B. Calderwood. 1997a. Protective immunity againstClostridium difficile toxin A induced by oral immunization with a live,attenuated Vibrio cholerae vector strain. Infect Immun 65: 2941-2949.

[0450] Ryan, E. T., J. R. Butterton, T. Zhang, M. A. Baker, S. L. J.Stanley, and S. B. Calderwood. 1997b. Oral immunization with attenuatedvaccine strains of Vibrio cholerae expressing a dodecapeptide repeat ofthe serine-rich Entamoeba histolytica protein fused to the cholera toxinB subunit induces systemic and mucosal antiamebic and anti-V choleraeantibody responses in mice. Infect Immun 65: 3118-3125.

[0451] Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press.

[0452] Seizer, G., T. Som, T. ltoh, and J. Tomizawa. 1983. The origin ofreplication of plasmid p15A and comparative studies on the nucleotidesequences around the origin of related plasmids. Cell 32:119.

[0453] Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993.Molecular genetics of amino-glycoside resistance genes and familialrelationships of the aminoglycoside-modifying enzymes. Microbiol Rev 57:138-163.

[0454] Siegler, R. L. 1995. The hemolytic uremic syndrome. PediatricNephrology 42:1505.

[0455] Siegler, R. L., A. T. Pavia, R. D. Christofferson, and M. K.Milligan. 1994. A 20-year population-based study of postdiarrhealhemolytic uremic syndrome in Utah. Pediatrics 94:35.

[0456] Sixma, T. K., P. E. Stein, W. G. Hol, and R. J. Read. 1993.Comparison of the B-pentamers of heat-labile enterotoxin andverotoxin-1: two structures with remarkable similarity anddissimilarity. Biochemistry 32:191.

[0457] Srinivasan, J., S. A. Tinge, R. Wright, J. C. Herr, and R.Curtiss III. 1995. Oral immunization with attenuated Salmonellaexpressing human sperm antigen induces antibodies in serum and thereproductive tract. Biology of Reproduction 53:462.

[0458] Stein, P. E., A. Boodhoo, G. J. Tyrrell, J. L. Brunton, and R. J.Read. 1992. Crystal structure of the cell-binding B oligomer ofverotoxin-1 from E. coli. Nature 355:748.

[0459] Stoker, N. G., N. F. Fairweather, and B. G. Spratt. 1982.Versatile low-copy-number plasmid vectors for cloning in Escherichiacoli. Gene 18: 335-341.

[0460] Streatfield, S. J., M. Sandkvist, T. K. Sixma, M. Bagdasarian, W.G. Hol, and T. R. Hirst. 1992. Intermolecular interactions between the Aand B subunits of heat-labile enterotoxin from Escherichia coli promoteholotoxin assembly and stability in vivo. Proceedings of the NationalAcademy of Sciences USA 89:12140.

[0461] Strockbine, N. A., L. R. M. Marques, J. W. Newland, H. W. Smith,R. K. Holmes, and A. D. O'Brien. 1986. Two toxin-converting phages fromEscherichia coli O157: H7 strain 933 encode antigenically distincttoxins with similar biologic activities. Infection and Immunity 53:135.

[0462] Strockbine, N. A., M. P. Jackson, L. M. Sung, R. K. Holmes, andA. D. O'Brien. 1988. Cloning and sequencing of the genes for Shiga toxinfrom Shigella dysenteriae Type 1. Journal of Bacteriology 170:1116.

[0463] Strugnell, R. A., D. Maskell, N. F. Fairweather, D. Pickard, A.Cockayne, C. Penn, and G. Dougan. 1990. Stable expression of foreignantigens from the chromosome of Salmonella typhimurium vaccine strains.Gene 88: 57-63.

[0464] Summers, D. K. The Biology of Plasmids, 65-91, 1996.

[0465] Summers, D. K. 1998. Timing, self-control and sense of directionare the secrets of multicopy plasmid stability. Mol Microbiol 29:1137-1145.

[0466] Summers, D. K. and D. J. Sherratt. 1984. Multimerization of highcopy number plasmids causes instability: ColE1 encodes a determinantessential for plasmid monomerization and stability. Cell 36:1097.

[0467] Tacket, C. O., D. M. Hone, R. Curtiss III, S. M. Kelly, G.Losonsky, L. Guers, A. M. Harris, R. Edelman, and M. M. Levine. 1992.Comparison of the safety and immunogenicity of ΔaroCΔaroD and ΔcyaΔcrpSalmonella typhi strains in adult volunteers. Infection and Immunity60:536.

[0468] Tacket, C. O., M. Sztein, G. Losonsky, S. S. Wasserman, J. P.Nataro, R. Edelman, D. Pickard, G. Dougan, S. Chaffield, and M. M.Levine. 1997. Safety of live oral Salmonella typhi vaccine strains withdeletions in htrA and aroC aroD and immune responses in humans.Infection and Immunity 65:452-456.

[0469] Tacket, C. O., S. M. Kelley, F. Schodel, G. Losonsky, J. P.Nataro, R. Edelman, M. M. Levine, and R. Curtiss III. 1997. Safety andimmunogenicity in humans of an attenuated Salmonella typhi vaccinevector strain expressing plasmid-encoded hepatitis B antigens stabilizedby the Asd-balanced lethal vector system. Infection and Immunity65:3381-3385.

[0470] Takeda, Y. 1995. Shiga and Siga-like (Vero) toxins. In Bacterialtoxins and virulence factors in disease. J. Moss, B. Iglewski, M.Vaughan and A. Tu, eds. Marcel Dekker, Inc. New York, p. 313.

[0471] Tauxe, R. V. 1998. Public health perspective onimmunoprophylactic strategies for Escherichia coli O157:H7: who or whatwould we immunize? In Escherichia coli O157:H7 and other Shigatoxin-producing E. coli strains. J. B. Kaper and A. D. O'Brien, eds. ASMPress, Washington, D.C. In press.

[0472] Tesh, V. L., J. A. Burris, J. W. Owens, V. M. Gordon, E. A.Wadolkowski, A. D. O'Brien, and J. E. Samuel. 1993. Comparison of therelative toxicities of Shiga-like toxins type I and type II for mice.Infection and Immunity 61:3392.

[0473] Thisted, T., A. K. Nielsen, and K. Gerdes. 1994. Mechanism ofpost-segregational killing: translation of Hok,SrnB and Pnd mRNAs ofplasmids R1, F and R483 is activated by 3′-end processing. EMBO Journal13:1950.

[0474] Thisted, T., N. S. Sorensen, and K. Gerdes. 1995. Mechanism ofpost-segregational killing: secondary structure analysis of the entireHok mRNA from plasmid R1 suggests a fold-back structure that preventstranslation and antisense RNA binding. Journal of Molecular Biology247:859.

[0475] Thisted, T., N. S. Sorensen, E. G. Wagner, and K. Gerdes. 1994.Mechanism of post-segregational killing: Sok antisense RNA interactswith Hok mRNA via its 5′-end single-stranded leader and competes withthe 3′-end of Hok mRNA for binding to the mok translational initiationregion. EMBO Journal 13:1960.

[0476] Tinge, S. A. and R. Curtiss III. 1990. Conservation of Salmonellatyphimurium virulence plasmid maintenance regions among Salmonellaserovars as a basis for plasmid curing. Infection and Immunity 58:3084.

[0477] Tinge, S. A. and R. Curtiss III. 1990. Isolation of thereplication and partitioning regions of the Salmonella typhimuriumvirulence plasmid and stabilization of heterologous replicons. Journalof Bacteriology 172:5266.

[0478] Twigg, A. J., and D. Sherratt. 1980. Trans-complementablecopy-number mutants of plasmid ColE1. Nature 283: 216-218.

[0479] Umbarger, H. E. 1978. Amino acid biosynthesis and its regulation.Annual Reviews in Biochemistry 47:533.

[0480] Valdivia, R. H. and S. Falkow. 1997. Fluorescence-based isolationof bacterial genes expressed within host cells. Science 277:2007.

[0481] Valdivia, R. H., A. E. Hromockyj, D. Monack, L. Ramakrishnan, andS. Falkow. 1996. Applications for green fluorescent protein (GFP) in thestudy of host-pathogen interactions. Gene 173:47.

[0482] Van Melderen, L., P. Bernard, and M. Couturier. 1994.Lon-dependent proteolysis of CcdA is the key control for activation ofCcdB in plasmid-free segregant bacteria. Mol Microbiol 11: 1151-1157.

[0483] Vicari, G., A. J. Olitzki, and Z. Olitzki. 1960. The action ofthe thermolabile toxin of Shigella dysenteriae on cells cultivated invitro. British Journal of Experimental Pathology 41:179.

[0484] Wada, K., Y. Wada, F. Ishibashi, T. Gojobori, and T. Ikemura.1992. Codon usage tabulated from the GenBank genetic sequence data.Nucleic Acids Research 20:2111.

[0485] Wadolkowski, E. A., L. M. Sung, J. A. Burris, J. E. Samuel, andA. D. O'Brien. 1990. Acute renal tubular necrosis and death of miceorally infected with Escherichia coli strains that produce Shiga-liketoxin type II. Infection and Immunity 58:3959.

[0486] Wahle, E., and A. Kornberg. 1988. The partition locus of plasmidpSC101 is a specific binding site for DNA gyrase. EMBO J 7: 1889-1895.

[0487] Wang, S. and T. Hazelrigg. 1994. Implications for bcd mRNAlocalization from spatial distribution of exu protein in Drosophilaoogenesis. Nature 369:400.

[0488] Wang, Y., Z. Zhang, S. Yang, and R. Wu. 1992. Cloning of parregion and the effect of par region on the stability of pUC9. ChineseJournal of Biotechnology 8:107.

[0489] Williams, K. R., J. B. Murphy, and J. W. Chase. 1984.Characterization of the structural and functional defect in theEscherichia coli single-stranded DNA binding protein encoded by thessb-1 mutant gene. Journal of Biological Chemistry 259:11804.

[0490] Wu, K., and T. K. Wood. 1994. Evaluation of the hokisok killerlocus for enhanced plasmid stability. Biotechnol Bioeng 44: 912-921.

[0491] Yamasaki, S., M. Furutani, K. Ito, K. Igarashi, M. Nishibuchi,and Y. Takeda. 1991. Importance of arginine at postion 170 of the Asubunit of Vero toxin 1 produced by enterohemorrhagic Escherichia colifor toxin activity. Microbial Pathogenesis 11:1.

[0492] Yanofsky, C., T. Platt, I. P. Crawford, B. P. Nichols, G. E.Christie, H. Horowitz, M. Van Cleemput, and A. M. Wu. 1981. The completenucleotide sequence of the tryptophan operon of Escherichia coli.Nucleic Acids Res 9: 6647-6668.

[0493] Yu, J. and J. B. Kaper. 1992. Cloning and characterization of theeae gene of enterohaemorrhagic Escherichia coli. Molecular Microbiology6:411.

[0494] Zalkin, H. and P. Nygaard. 1996. Biosynthesis of purinenucleotides. In Escherichia coli and Salmonella: Cellular and molecularbiology. 2nd ed. F. C. Neidhardt, J. L. Ingraham, E. C. C. Lin, K. B.Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.Umbarger, eds. ASM Press, Washington, D.C. p. 561.

[0495] Zhang, X., Y. Lou, M. Koopman, T. Doggett, K. S. K. Tung, and R.Curtiss III. 1997. Antibody responses and infertility in mice followingoral immunization with attenuated Salmonella typhimurium expressingrecombinant murine ZP3. Biology of Reproduction 56:33.

[0496] Zoja, C., D. Corna, C. Farina, G. Sacchi, C. A. Lingwood, M. P.Doyle, V. V. Padhye, M. Abbate, and G. Remuzzi. 1992. Verotoxinglycolipid receptors determine the localization of microangiopathicprocess in rabbits given verotoxin-1. Journal of Laboratory and ClinicalMedicine 120:229.

[0497] Zurita, M., F. Bolivar, and X. Soberon. 1984. Construction andcharacterization of new cloning vehicles. VII. Construction of plasmidpBR327par, a completely sequenced, stable derivative of pBR327containing the par locus of pSC101. Gene 28:119.

1 40 1 4196 DNA Artificial Sequence Complete nucleotide sequence ofpGEN2 1 gaattctgtg gtagcacaga ataatgaaaa gtgtgtaaag aagggtaaaaaaaaccgaat 60 gcgaggcatc cggttgaaat aggggtaaac agacattcag aaatgaatgacggtaataaa 120 taaagttaat gatgatagcg ggagttattc tagttgcgag tgaaggttttgttttgacat 180 tcagtgctgt caaatactta agaataagtt attgatttta accttgaattattattgctt 240 gatgttaggt gcttatttcg ccattccgca ataatcttaa aaagttcccttgcatttaca 300 ttttgaaaca tctatagcga taaatgaaac atcttaaaag ttttagtatcatattcgtgt 360 tggattattc tgcatttttg gggagaatgg acttgccgac tgattaatgagggttaatca 420 gtatgcagtg gcataaaaaa gcaaataaag gcatataaca gatcgatcttaaacatccac 480 aggaggatat ctgatgagta aaggagaaga acttttcact ggagttgtcccaattcttgt 540 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagggtgaaggtga 600 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaactacctgttcc 660 atggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcccgttatccgga 720 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatgtacaggaacg 780 cactatatct ttcaaagatg acgggaacta caagacgcgt gctgaagtcaagtttgaagg 840 tgataccctt gttaatcgta tcgagttaaa aggtattgat tttaaagaagatggaaacat 900 tctcggacac aaactcgagt acaactataa ctcacacaat gtatacatcacggcagacaa 960 acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacattgaagatggatccgt 1020 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctgtccttttacc 1080 agacaaccat tacctgtcga cacaatctgc cctttcgaaa gatcccaacgaaaagcgtga 1140 ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggcatggatgagct 1200 ctacaaataa tgagctagcc cgcctaatga gcgggctttt ttttctcggcctagggccag 1260 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccataggctccgcccc 1320 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaacccgacaggacta 1380 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgttccgaccctg 1440 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgctttctcatagc 1500 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctgggctgtgtgcac 1560 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtcttgagtccaac 1620 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggattagcagagcg 1680 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacggctacactaga 1740 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaaaagagttggt 1800 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgtttgcaagcag 1860 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttctacggggtct 1920 gacgctcagt agatctaaaa cactaggccc aagagtttgt agaaacgcaaaaaggccatc 1980 cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggcgtcctgcccg 2040 ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggatttgtcctact 2100 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctttcgactgagc 2160 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagaccccacactac 2220 catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtgggaccaccgcgc 2280 tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgatttaatctgta 2340 tcaggctgaa aatcttctct catccgccaa aacagccaag ctggatccccgatcttatca 2400 ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcagacaaggtatag 2460 ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcggcataaatcgc 2520 cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcgagcgatccttg 2580 aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgcaacgcgggcat 2640 cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagcctcgcgtcgc 2700 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataatggcctgctt 2760 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgagggcgtgcaagat 2820 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagcggtcctcgcc 2880 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataaagaagacagt 2940 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctgactgggttgaa 3000 ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcattaggaagcagcc 3060 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcatgcaaggagat 3120 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccgaaacaagcgct 3180 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcgatataggcgcc 3240 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgtagaggatcca 3300 caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg gctccaagtagcgaagcgag 3360 caggactggg cggcggccaa agcggtcgga cagtgctccg agaacgggtgcgcatagaaa 3420 ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgctgtcggaatg 3480 gacgatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgcctacagcatc 3540 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatttttttttcctc 3600 cttattttct agacaacatc agcaaggaga aaggggctac cggcgaaccagcagcccctt 3660 tataaaggcg cttcagtagt cagaccagca tcagtcctga aaaggcgggcctgcgcccgc 3720 ctccaggttg ctacttaccg gattcgtaag ccatgaaagc cgccacctccctgtgtccgt 3780 ctctgtaacg aatctcgcac agcgattttc gtgtcagata agtgaatatcaacagtgtga 3840 gacacacgat caacacacac cagacaaggg aacttcgtgg tagtttcatggccttcttct 3900 ccttgcgcaa agcgcggtaa gaggctatcc tgatgtggac tagacatagggatgcctcgt 3960 ggtggttaat gaaaattaac ttactacggg gctatcttct ttctgccacacaacacggca 4020 acaaaccacc ttcacgtcat gaggcagaaa gcctcaagcg ccgggcacatcatagcccat 4080 atacctgcac gctgaccaca ctcactttcc ctgaaaataa tccgctcattcagaccgttc 4140 acgggaaatc cgtgtgattg ttgccgcatc acgctgcctc ccggagtttgtctcga 4196 2 1197 DNA Artificial Sequence Partial nucleotide sequenceof pGEN3 nucleotides 1201-2397 encoding ori15A 2 ctacaaataa tgagctagcccgcctaatga gcgggctttt ttttctcggc ctaggagata 60 cttaacaggg aagtgagagggccgcggcaa agccgttttt ccataggctc cgcccccctg 120 acaagcatca cgaaatctgacgctcaaatc agtggtggcg aaacccgaca ggactataaa 180 gataccaggc gtttccccctggcggctccc tcgtgcgctc tcctgttcct gcctttcggt 240 ttaccggtgt cattccgctgttatggccgc gtttgtctca ttccacgcct gacactcagt 300 tccgggtagg cagttcgctccaagctggac tgtatgcacg aaccccccgt tcagtccgac 360 cgctgcgcct tatccggtaactatcgtctt gagtccaacc cggaaagaca tgcaaaagca 420 ccactggcag cagccactggtaattgattt agaggagtta gtcttgaagt catgcgccgg 480 ttaaggctaa actgaaaggacaagttttgg tgactgcgct cctccaagcc agttacctcg 540 gttcaaagag ttggtagctcagagaacctt cgaaaaaccg ccctgcaagg cggttttttc 600 gttttcagag caagagattacgcgcagacc aaaacgatct caagaagatc atcttattaa 660 tcagataaaa tatttctaggatctaaaaca ctaggcccaa gagtttgtag aaacgcaaaa 720 aggccatccg tcaggatggccttctgctta atttgatgcc tggcagttta tggcgggcgt 780 cctgcccgcc accctccgggccgttgcttc gcaacgttca aatccgctcc cggcggattt 840 gtcctactca ggagagcgttcaccgacaaa caacagataa aacgaaaggc ccagtctttc 900 gactgagcct ttcgttttatttgatgcctg gcagttccct actctcgcat ggggagaccc 960 cacactacca tcggcgctacggcgtttcac ttctgagttc ggcatggggt caggtgggac 1020 caccgcgcta ctgccgccaggcaaattctg ttttatcaga ccgcttctgc gttctgattt 1080 aatctgtatc aggctgaaaatcttctctca tccgccaaaa cagccaagct ggatccccga 1140 tcttatcagg tcgaggtggcccggctccat gcaccgcgac gcaacgcggg gaggcag 1197 3 2647 DNA ArtificialSequence Partial nucleotide sequence of pGEN4 nucleotides 1201-3848encoding ori101 3 ctacaaataa tgagctagcc cgcctaatga gcgggctttt ttttctcggcctaggtttca 60 cctgttctat taggtgttac atgctgttca tctgttacat tgtcgatctgttcatggtga 120 acagctttaa atgcaccaaa aactcgtaaa agctctgatg tatctatcttttttacaccg 180 ttttcatctg tgcatatgga cagttttccc tttgatatct aacggtgaacagttgttcta 240 cttttgtttg ttagtcttga tgcttcactg atagatacaa gagccataagaacctcagat 300 ccttccgtat ttagccagta tgttctctag tgtggttcgt tgtttttgcgtgagccatga 360 gaacgaacca ttgagatcat gcttactttg catgtcactc aaaaattttgcctcaaaact 420 ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt tttcttagtccgttacgtag 480 gtaggaatct gatgtaatgg ttgttggtat tttgtcacca ttcatttttatctggttgtt 540 ctcaagttcg gttacgagat ccatttgtct atctagttca acttggaaaatcaacgtatc 600 agtcgggcgg cctcgcttat caaccaccaa tttcatattg ctgtaagtgtttaaatcttt 660 acttattggt ttcaaaaccc attggttaag ccttttaaac tcatggtagttattttcaag 720 cattaacatg aacttaaatt catcaaggct aatctctata tttgccttgtgagttttctt 780 ttgtgttagt tcttttaata accactcata aatcctcata gagtatttgttttcaaaaga 840 cttaacatgt tccagattat attttatgaa tttttttaac tggaaaagataaggcaatat 900 ctcttcacta aaaactaatt ctaatttttc gcttgagaac ttggcatagtttgtccactg 960 gaaaatctca aagcctttaa ccaaaggatt cctgatttcc acagttctcgtcatcagctc 1020 tctggttgct ttagctaata caccataagc attttcccta ctgatgttcatcatctgagc 1080 gtattggtta taagtgaacg ataccgtccg ttctttcctt gtagggttttcaatcgtggg 1140 gttgagtagt gccacacagc ataaaattag cttggtttca tgctccgttaagtcatagcg 1200 actaatcgct agttcatttg ctttgaaaac aactaattca gacatacatctcaattggtc 1260 taggtgattt taatcactat accaattgag atgggctagt caatgataattactagtcct 1320 tttcctttga gttgtgggta tctgtaaatt ctgctagacc tttgctggaaaacttgtaaa 1380 ttctgctaga ccctctgtaa attccgctag acctttgtgt gttttttttgtttatattca 1440 agtggttata atttatagaa taaagaaaga ataaaaaaag ataaaaagaatagatcccag 1500 ccctgtgtat aactcactac tttagtcagt tccgcagtat tacaaaaggatgtcgcaaac 1560 gctgtttgct cctctacaaa acagacctta aaaccctaaa ggcttaagtagcaccctcgc 1620 aagctcgggc aaatcgctga atattccttt tgtctccgac catcaggcacctgagtcgct 1680 gtctttttcg tgacattcag ttcgctgcgc tcacggctct ggcagtgaatgggggtaaat 1740 ggcactacag gcgcctttta tggattcatg caaggaaact acccataatacaagaaaagc 1800 ccgtcacggg cttctcaggg cgttttatgg cgggtctgct atgtggtgctatctgacttt 1860 ttgctgttca gcagttcctg ccctctgatt ttccagtctg accacttcggattatcccgt 1920 gacaggtcat tcagactggc taatgcaccc agtaaggcag cggtatcatcaacaggctta 1980 cccgtcttac tgtcaaccgg atctaaaaca ctaggcccaa gagtttgtagaaacgcaaaa 2040 aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagtttatggcgggcgt 2100 cctgcccgcc accctccggg ccgttgcttc gcaacgttca aatccgctcccggcggattt 2160 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggcccagtctttc 2220 gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcatggggagaccc 2280 cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggtcaggtgggac 2340 caccgcgcta ctgccgccag gcaaattctg ttttatcaga ccgcttctgcgttctgattt 2400 aatctgtatc aggctgaaaa tcttctctca tccgccaaaa cagccaagctggatccccga 2460 tcttatcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggggaggcagaca 2520 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgccgaggcggca 2580 taaatcgccg tgacgatcag cggtccagtg atcgaagtta ggctggtaagagccgcgagc 2640 gatcctt 2647 4 41 DNA Artificial Sequence Portion ofpromoter sequence 4 catataacag atcttaatca tccacaggag gatatctgat g 41 545 DNA Artificial Sequence Portion of promoter sequence 5 catataacagatcgatctta aacatccaca ggaggatatc tgatg 45 6 66 DNA Artificial SequencePrimer 6 gaattcgcgc gcttcgcgat tcagtcgcgt tccttcacag ctggcgcaggggcgattact 60 gatgaa 66 7 66 DNA Artificial Sequence Primer 7 cccgggagtctcctgaatac gtttcataaa tagtgtaaac gcgtgagtgt accatttcca 60 cgtagc 66 8 69DNA Artificial Sequence Primer 8 cccgggtaaa aaactcaaag cgttatttgcattttcgcta tagttctcgt ctgctgaaat 60 gcctggtgt 69 9 77 DNA ArtificialSequence Primer 9 gaattccatt tctatcaata aattactatt agttttgtct tctaaccaagcctctatttt 60 atgagtatcc tcttcag 77 10 72 DNA Artificial Sequence Primer10 gctagcatgg ccagcagagg cgtaaacaag gtgattctcg ttggtaatct gggccaggac 60ccggaagtac gc 72 11 64 DNA Artificial Sequence Primer 11 gctagctcagaacggaatgt cgtcgtcaaa atccattggc ggttcgttag acggcgctgg 60 cgcg 64 12 45DNA Artificial Sequence Primer 12 gcaggaaaga acatgtgagc ctagggccagcaaaaggcca ggaac 45 13 53 DNA Artificial Sequence Primer 13 catgaccaaaatcccttaac tagtgtttta gatctactga gcgtcagacc ccg 53 14 53 DNA ArtificialSequence Primer 14 cggggtctga cgctcagtag atctaaaaca ctagttaagggattttggtc atg 53 15 49 DNA Artificial Sequence Primer 15 gctgtcaaacatgagaattc tagaagacga aagggcctcg tgatacgcc 49 16 64 DNA ArtificialSequence Primer 16 acagcctgca gacagatctt gacagctgga tcgcactctggtataattgg gaagccctgc 60 aaag 64 17 59 DNA Artificial Sequence Primer 17cgaagcccaa cctttcatag aagctagcgg tggatccgaa atctcgtgat ggcaggttg 59 1830 DNA Artificial Sequence Primer 18 aacaagcgtt ataggaattc tgtggtagca 3019 30 DNA Artificial Sequence Primer 19 actttcatgt tattaaagat ctgttatatg30 20 59 DNA Artificial Sequence Primer 20 agatcttaat catccacaggaggctttctg atgagtaaag gagaagaact tttcactgg 59 21 32 DNA ArtificialSequence Primer 21 gctagctcat tatttgtaga gctcatccat gc 32 22 54 DNAArtificial Sequence Primer 22 agatctgaat tctagatcat gtttgacagcttatcatcga taagctttaa tgcg 54 23 51 DNA Artificial Sequence Primer 23agatcttatc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc g 51 24 61 DNAArtificial Sequence Primer 24 cgcgaattct cgagacaaac tccgggaggcagcgtgatgc ggcaacaatc acacggattt 60 c 61 25 69 DNA Artificial SequencePrimer 25 atgagcgcat tgttagattt catttttttt tcctccttat tttctagacaacatcagcaa 60 ggagaaagg 69 26 69 DNA Artificial Sequence Primer 26cctttctcct tgctgatgtt gtctagaaaa taaggaggaa aaaaaaatga aatctaacaa 60tgcgctcat 69 27 65 DNA Artificial Sequence Primer 27 gctacatttgaagagataaa ttgcactgga tcctagaaat attttatctg attaataaga 60 tgatc 65 28 50DNA Artificial Sequence Primer 28 cggagatttc ctggaagatg cctaggagatacttaacagg gaagtgagag 50 29 61 DNA Artificial Sequence Primer 29gtctgccgga ttgcttatcc tggcggatcc ggttgacagt aagacgggta agcctgttga 60 t61 30 61 DNA Artificial Sequence Primer 30 cctaggtttc acctgttctattaggtgtta catgctgttc atctgttaca ttgtcgatct 60 g 61 31 61 DNA ArtificialSequence Primer 31 aggcttaagt agcaccctcg caagatctgg caaatcgctgaatattcctt ttgtctccga 60 c 61 32 70 DNA Artificial Sequence Primer 32gagggcgccc cagctggcaa ttctagactc gagcactttt gttacccgcc aaacaaaacc 60caaaaacaac 70 33 61 DNA Artificial Sequence Primer 33 agaagaaaaatcgaattcca gcatgaagag tttcagaaaa tgacagagcg tgagcaagtg 60 c 61 34 59 DNAArtificial Sequence Primer 34 cgaagcccaa cctttcatag aaactagtggtggaatcgaa atctcgtgat ggcaggttg 59 35 70 DNA Artificial Sequence Primer35 gttgtttttg ggttttgttt ggcgggtaac aaaagtgctc gagtctagaa ttgccagctg 60gggcgccctc 70 36 34 DNA Artificial Sequence Modified Promoter Sequence36 agatcnntaa ncatccacag gaggatatct gatg 34 37 15 DNA ArtificialSequence Modified Shiga toxin sequence 37 acagcagacg cgtta 15 38 15 DNAArtificial Sequence Modified Shiga toxin sequence 38 ctgaacctag ggcga 1539 15 DNA Artificial Sequence Modified Shiga toxin sequence 39gaattcgcga ccagt 15 40 15 DNA Artificial Sequence Modified Shiga toxinsequence 40 gaatcagatt ctgga 15

What is claimed is:
 1. An independently functioning expression cassettecomprising a nucleotide sequence encoding: (a) an origin of replication;and (b) a nucleotide sequence encoding a plasmid maintenance systemcomprising: (i) at least one post-segregational killing function; and(ii) at least one partitioning function.
 2. The independentlyfunctioning expression cassette of claim 1 wherein the origin ofreplication is selected from the group consisting of: oriE1, ori101,ori15A and derivatives thereof.
 3. The independently functioningexpression cassette of claim 1 wherein the post-segregational killingfunction is selected from the group consisting of asd, ssb, phd-doc andhok-sok.
 4. The independently functioning expression cassette of claim 1wherein the post-segregational killing function is a substantialhomologue of a naturally-occuring post-segregational killing function.5. The independently functioning expression cassette of claim 1 whereinthe partitioning function comprises an active partitioning function. 6.The independently functioning expression cassette of claim 1 wherein thepartitioning function comprises a passive partitioning function.
 7. Theindependently functioning expression cassette of claim 1 wherein thepartitioning function is the par locus of pSC101.
 8. The independentlyfunctioning expression cassette of claim 1 wherein the partitioningfunction comprises parA.
 9. The independently functioning expressioncassette of claim 1 wherein the partitioning function is a substantialhomologue of a naturally-occurring partitioning function.
 10. Anamplifiable plasmid repticon comprising the independently functioningexpression cassette of claim
 1. 11. The amplifiable plasmid replicon ofclaim 10 further comprising independently functioning expressioncassette comprising a nucleotide sequence encoding an antigen ofinterest transcriptionally controlled by a promoter.
 12. The amplifiableplasmid replicon of claim 11 having a copy number that can be controlledto vary from 0 copies per cell to greater than 75 copies per cell andwherein the antigen of interest is a regulated test antigen forexpression in a bacterium such that the inducible promoter is positionedto control expression of the nucleotide sequence, such that as inductionof the promoter is increased, test antigen expression is increased, andthe metabolic burden of the bacterium is increased.
 13. A bacterial cellcomprising the amplifiable plasmid replicon of claim
 12. 14. Theamplifiable plasmid replicon of claim 11 wherein the promoter is derivedfrom the ompC promoter.
 15. The amplifiable plasmid replicon of claim 11wherein the promoter is the ompC promoter.
 16. The amplifiable plasmidreplicon of claim 11 wherein the promoter is the ompC promoter fragmentfrom E. coli spanning nucleotides +70 through −389.
 17. The amplifiableplasmid replicon of claim 11 wherein the promoter is a modified ompCpromoter characterized in that said modified ompC promoter exhibitshigher rates of osmotically regulated expression in relation to acorresponding ompC promoter without such point mutations.
 18. Theamplifiable plasmid replicon of claim 17 wherein the modified ompCpromoter comprises a modified BglII site.
 19. The ampliflable plasmidreplicon of claim 17 wherein the modified ompC promoter is without aBglII site.
 20. The amplifiable plasmid replicon of claim 18 wherein themodified ompC promoter comprises the following sequence 5′ to 3′beginning with the modified BglII site and ending with the ATG startcodon: AGATCX¹X²TAAX³CATCCACAGGAGGATATCTGATG, wherein: (a) X¹ isselected from the group consisting of G, C and A; (b) X² is an inserthaving from 1 to 5 nucleotides; (c) X³ is selected from the groupconsisting of A, T, G and C.
 21. The amplifiable plasmid replicon ofclaim 18 wherein X¹ is G.
 22. The amplifiable plasmid replicon of claim18 wherein X² has from 1 to 4 nucleotides.
 23. The amplifiable plasmidreplicon of claim 18 wherein X² has 4 nucleotides.
 24. The amplifiableplasmid replicon of claim 18 wherein X² has 4 nucleotides, independentlyselected from the group consisting of A, T and C.
 25. The amplifiableplasmid replicon of claim 18 wherein X² comprises a nucleotide ornucleotide sequence selected from the group consisting of ATCT; ATC; AT;TCT; CT; TC; A; T; C; and T.
 26. The amplifiable plasmid replicon ofclaim 18 wherein X² is selected from the group consisting of ATCT; ATC;AT; TCT; CT; TC; A; T; C; and T.
 27. The amplifiable plasmid replicon ofclaim 18 wherein X² is ATCT.
 28. The amplifiable plasmid replicon ofclaim 18 wherein X³ is A.
 29. The amplifiable plasmid replicon of claim11 wherein the antigen of interest comprises the green fluorescentprotein or a functional equivalent thereof.
 30. The amplifiable plasmidreplicon of claim 11 wherein the antigen of interest comprises adetoxified Shiga toxin and/or a substantial homologue thereof.
 31. Theamplifiable plasmid replicon of claim 11 wherein the antigen of interestcomprises a Shiga toxin 2 antigen selected from the group comprising of:Shiga toxin 2 B subunit pentamers and genetically detoxified Shiga toxin2 (Stx 2).
 32. The amplifiable plasmid replicon of claim 30 wherein thegene encoding the detoxified Shiga toxin 2 has modified segmentsselected from the group consisting of: (797)-  ACA GCA GAG GCG TTA -(811); (902)-  CTG AAC CTA GGG CGA   (916); (1345)- GAA TTC GCG ACCAGT - (1359); and (1435)- GAA TCA GAT TCT GGA - (1449).


33. A bacterial cell comprising the amplifiable plasmid replicon ofclaim
 11. 34. The amplifiable plasmid replicon of claim 10 furthercomprising an independently functioning expression cassette comprising anucleotide sequence encoding a selectable marker.
 35. The amplifiableplasmid replicon of claim 34 wherein the selectable marker does notconfer resistance to any antibiotic which is ordinarily used in medicaltreatment of humans.
 36. The amplifiable plasmid replicon of claim 34wherein the selectable marker comprises β-lactamase and/or a functionalequivalent thereof.
 37. The amplifiable plasmid replicon of claim 34wherein the nucleotide sequence encoding the resistance marker isselected from the group consisting of tetA, bla and functionalequivalents thereof.
 38. A cell comprising the amplifiable plasmidreplicon of claim
 34. 39. A bacterial cell comprising the amplifiableplasmid replicon of claim
 34. 40. An attenuated bacterial vector vaccinecomprising a bacterial species containing a replicon, said repliconcomprising: (a) a nucleotide sequence encoding an antigen of interest;and (b) a nucleotide sequence encoding a plasmid maintenance system. 41.The bacterial vector vaccine of claim 40 wherein the nucleotide sequenceencoding the antigen of interest is contained in an independentlyfunctioning genetic cassette.
 42. The bacterial vector vaccine of claim40 wherein the nucleotide sequence encoding the plasmid maintenancesystem is contained within independently functioning genetic cassettes.43. The attenuated bacterial live vector vaccine of claim 40 wherein thebacterial species is Salmonella typhi.
 44. The attenuated bacterial livevector vaccine of claim 40 wherein the replicon further comprises anompC promoter, or a substantial homologue thereof, controllingexpression of the antigen of interest.
 45. The attenuated bacterial livevector vaccine of claim 40 wherein the replicon further comprises amodified ompC promoter controlling expression of the antigen ofinterest, wherein the modified ompC promoter exhibits higher rates ofosmotically controlled expression as compared to a non-modified ompCpromoter.
 46. The attenuated bacterial live vector vaccine of claim 45wherein the modified ompC promoter comprises a modified BglII site. 47.The attenuated bacterial live vector vaccine of claim 45 wherein themodified ompC promoter is without a complete BglII site.
 48. Theattenuated bacterial live vector vaccine of claim 46 wherein themodified ompC promoter comprises the following sequence 5′ to 3′beginning with the modified BglII site and ending with the ATG startcodon: AGATCX¹X²TAAX³CATCCACAGGAGGATATCTGATG, wherein: (a) X¹ isselected from the group consisting of G, C and A; (b) X² is optionallypresent and is an insert having from 1 to 5 nucleotides; (c) X³ isselected from the group consisting of A, T, G and C.
 49. The attenuatedbacterial live vector vaccine of claim 48 wherein X¹ is G.
 50. Theattenuated bacterial live vector vaccine of claim 48 wherein X² has from1 to 4 nucleotides.
 51. The attenuated bacterial live vector vaccine ofclaim 48 wherein X² has 4 nucleotides.
 52. The attenuated bacterial livevector vaccine of claim 48 wherein X² has 4 nucleotides, independentlyselected from the group consisting of A, T and C.
 53. The attenuatedbacterial live vector vaccine of claim 48 wherein X² comprises anucleotide or nucleotide sequence selected from the group consisting ofATCT; ATC; AT; TCT; CT; TC; A; T; C; and T.
 54. The attenuated bacteriallive vector vaccine of claim 48 wherein X² is selected from the groupconsisting of ATCT; ATC; AT; TCT; CT; TC; A; T; C; and T.
 55. Theattenuated bacterial live vector vaccine of claim 48 wherein X² is ATCT.56. The attenuated bacterial live vector vaccine of claim 48 wherein X³is A.
 57. The attenuated bacterial live vector vaccine of claim 40wherein the plasmid maintenance system comprises: (a) at least onepost-segregational killing function; and (b) at least one partitioningfunction.
 58. The attenuated bacterial live vector vaccine of claim 57wherein the post-segregational killing function is selected from thegroup consisting of balanced lethal functions, proteic functions andantisense functions.
 59. The attenuated bacterial live vector vaccine ofclaim 57 wherein the post-segregational killing function is selectedfrom the group consisting of asd, ssb, phd-doc, hok-sok, and substantialhomologues thereof.
 60. The attenuated bacterial live vector vaccine ofclaim 57 wherein the partitioning function comprises an activepartitioning function.
 61. The attenuated bacterial live vector vaccineof claim 57 wherein the partitioning function comprises a passivepartitioning function.
 62. The attenuated bacterial live vector vaccineof claim 57 wherein the partitioning function comprises the par locus ofpSC101 and/or a substantial homologue thereof.
 63. The attenuatedbacterial live vector vaccine of claim 57 wherein the partitioningfunction comprises parA and/or a substantial homologue thereof.
 64. Theattenuated bacterial live vector vaccine of claim 40 wherein the antigenof interest is a test antigen.
 65. The attenuated bacterial live vectorvaccine of claim 64 wherein the test antigen is selected from the groupconsisting of green fluorescent protein, functional equivalents of greenfluorescent protein and substantial homologues of green fluorescentprotein.
 66. The attenuated bacterial live vector vaccine of claim 40wherein the antigen of interest is a detoxified Shiga toxin.
 67. Theattenuated bacterial live vector vaccine of claim 40 wherein the antigenis one or more Shiga toxin 2 antigens selected from the group comprisingShiga toxin 2 B subunit pentamers and a genetically detoxified Stx 2.68. The attenuated bacterial live vector vaccine of claim 67 wherein thegene encoding the detoxified Shiga toxin 2 has mutations selected fromthe group consisting of: (797)  - ACA GCA GAC GCG TTA - (811); (902)  -CTG AAC CTA GGG CGA - (916); (1345) - GAA TTC GCG ACC AGT - (1359); and(1435) - GAA TCA GAT TCT GGA - (1449).


69. The attenuated bacterial live vector vaccine of claim 40 wherein thereplicon further comprises a nucleotide sequence encoding a resistancemarker.
 70. The attenuated bacterial live vector vaccine of claim 69wherein the nucleotide sequence encoding the resistance marker iscontained in an independently functioning genetic cassette.
 71. Theattenuated bacterial live vector vaccine of claim 69 wherein theselectable marker does not confer resistance to any antibiotic which isordinarily used in medical treatment of humans
 72. The attenuatedbacterial live vector vaccine of claim 69 wherein the nucleotidesequence encoding the resistance marker comprises bla and/or asubstantial homologue thereof.
 73. The attenuated bacterial live vectorvaccine of claim 69 wherein the nucleotide sequence encoding theresistance marker comprises tetA and/or a substantial homologue thereof.74. A conditionally unstable plasmid for examining changes in plasmidstability resulting from incorporation of plasmid maintenance systems,said plasmid comprising an origin of replication yielding an averagecopy number which falls within the range of from about 2 to about 75copies and a promoter driving the expression of a protein or peptide,overexpression of which imposes a metabolic burden on a bacterium, whichfavors plasmid loss.
 75. The conditionally unstable plasmid of claim 74wherein the average copy number falls within the range of about 5 toabout 60 copies.
 76. The conditionally unstable plasmid of claim 74wherein the promoter comprised an ompC promoter or a substantialhomologue thereof.
 77. The conditionally unstable plasmid of claim 74wherein the protein or peptide is selected from the group consisting ofgreen fluorescent protein, functional equivalents of green fluorescentprotein and substantial homologues of green fluorescent protein
 78. Theconditionally unstable plasmid of claim 74, wherein the average copynumber is selected from the group consisting of: about 5 copies percell; about 15 copies per cell; and about 60 copies per cell.
 79. Theconditionally unstable plasmid of claim 74 wherein the origin ofreplication is selected from the group consisting of the origin ofreplication of plasmid pSC101, origin of replication of plasmidpACYC184, origin of replication of plasmid pAT153, and substantialhomologues of any of such origins of replication.
 80. The conditionallyunstable plasmid of claim 74 wherein the origin of replication is frompSC101, conferring a copy number of approximately 5 copies per genomeequivalent.
 81. The conditionally unstable plasmid of claim 74 whereinthe origin of replication is from pACYC184, conferring a copy number ofapproximately 15 copies per genome equivalent.
 82. The conditionallyunstable plasmid of claim 74 wherein the origin of replication is frompAT153, conferring a copy number of approximately 60 copies per genomeequivalent.
 83. A method for eliciting an immune response in a subjectcomprising administering to the subject a bacterial live vector vaccinecomprising a bacterial strain comprising an expression vectorcomprising: (a) a nucleotide sequence encoding an antigen; (b) apromoter controlling expression of the antigen; and (c) a nucleotidesequence encoding at least one plasmid maintenance system.
 84. Themethod of claim 83 wherein the bacterial live vector vaccine isadministered in an immunizingly effective amount.
 85. The method ofclaim 83 wherein the bacterial live vector is an attenuated Salmonellatyphi species.
 86. The method of claim 83 wherein (a) and (b) arecontained in an independently functioning genetic cassette.
 87. Themethod of claim 83 wherein (c) is contained within independentlyfunctioning genetic cassettes.
 88. The method of claim 83 wherein thepromoter is an inducible promoter.
 89. The method of claim 83 whereinthe promoter is an ompC promoter or a functional equivalent thereof. 90.The method of claim 83 wherein the promoter is a modified ompC promoter,phenotypically characterized in that said promoter exhibits higher ratesof osmotically controlled expression in relationship to a correspondingompC promoter without such point mutations.
 91. The method of claim 90wherein the modified ompC promoter comprises a modified BglII site, oris without a complete BglII site.
 92. The method of claim 91 wherein themodified ompC promoter comprises the following sequence 5′ to 3′beginning with the modified BglII site and ending with the ATG startcodon: AGATCX¹X²TAAX³CATCCACAGGAGGATATCTGATG, wherein: (a) X¹ isselected from the group consisting of G, C and A; (b) X² is an inserthaving from 1 to 5 nucleotides; (c) X³ is selected from the groupconsisting of A, T, G and C.
 93. The method of claim 92 wherein X¹ is G.94. The method of claim 92 wherein X² has from 1 to 4 nucleotides. 95.The method of claim 92 wherein X² has 4 nucleotides.
 96. The method ofclaim 92 wherein X² has 4 nucleotides, independently selected from thegroup consisting of A, T and C.
 97. The method of claim 92 wherein X²comprises a nucleotide or nucleotide sequence selected from the groupconsisting of ATCT; ATC; AT; TCT; CT; TC; A; T; C; and T.
 98. The methodof claim 92 wherein X² is selected from the group consisting of ATCT;ATC; AT; TCT; CT; TC; A; T; C; and T.
 99. The method of claim 92 whereinX² is ATCT.
 100. The method of claim 92 wherein X³ is A.
 101. The methodof claim 83 wherein the plasmid maintenance system comprises: (a) atleast one post-segregational killing function; and (b) at least onepartitioning plasmid function.
 102. The method of claim 101 wherein thepost-segregational killing function is contained in an independentlyfunctioning genetic cassette.
 103. The method of claim 101 wherein thepost-segregational killing function is selected from the groupconsisting of balanced lethal functions, proteic functions and antisensefunctions.
 104. The method of claim 101 wherein the post-segregationalkilling function is selected from the group consisting of asd, ssb,phd-doc, hok-sok and substantial homologues thereof.
 105. The method ofclaim 101 wherein the partitioning function is contained in anindependently functioning genetic cassette.
 106. The method of claim 101wherein the partitioning function comprises an active partitioningfunction.
 107. The method of claim 101 wherein the partitioning functioncomprises a passive partitioning function.
 108. The method of claim 101wherein the partitioning function comprises the par locus of pSC101and/or a substantial homologue thereof.
 109. The method of claim 101wherein the partitioning function comprises parA and/or a substantialhomologue thereof.
 110. The method of claim 101 wherein the antigen(s)comprise at least one genetically detoxified Shiga toxin.
 111. Themethod of claim 101 wherein the antigen(s) include at least one Shigatoxin 2 (Stx2) antigen selected from the group comprising Shiga toxin 2B subunit pentamers and a genetically detoxified Stx
 2. 112. The methodof claim 83 wherein the nucleotide sequence further comprises anucleotide sequence encoding a selectable marker which does not conferresistance to any antibiotic which is ordinarily used in the treatmentof humans.
 113. The method of claim 112 wherein the nucleotide sequenceencoding the selectable marker is contained in an independentlyfunctioning genetic cassette.
 114. The method of claim 83 wherein thesubject is a human.
 115. The method of claim 83 wherein the subject is abovine.
 116. A method of making a stabilized bacterial live vectorvaccine comprising transforming a bacterial live vector with a repliconcomprising: (a) a plasmid maintenance system comprising: (i) at leastone post-segregational killing function; and (ii) at least onepartitioning function; and (b) a nucleotide sequence encoding one ormore antigens.
 117. The method of claim 116 wherein the post segregationkilling function is contained in an independently functioning geneticcassette.
 118. The method of claim 116 wherein the partitioning functionis contained in an independently functioning genetic cassette.
 119. Themethod of claim 116 wherein the post-segregational killing function isselected from the group consisting of balanced lethal functions, proteicfunctions and antisense functions.
 120. The method of claim 116 whereinthe post-segregational killing function is selected from the groupconsisting of asd, ssb, phd-doc, hok-sok, and substantial homologuesthereof.
 121. The method of claim 116 wherein the partitioning functionis an active partitioning function.
 122. The method of claim 116 whereinthe partitioning function is a passive partitioning function
 123. Themethod of claim 116 wherein the partitioning function comprises the parlocus of pSC101 and/or a substantial homologue thereof.
 124. The methodof claim 116 wherein the partitioning function comprises parA and/or asubstantial homologue thereof.
 125. The method of claim 116 wherein theone or more antigens comprise at least one detoxified Shiga toxin. 126.The method of claim 116 wherein the one or more antigens comprise one ormore Shiga toxin 2 antigens selected from the group comprising Shigatoxin 2 B subunit pentamers and a detoxified Stx
 2. 127. The method ofclaim 125 wherein the gene encoding the detoxified Shiga toxin 2 hasmutations selected from the group consisting of: (797)  - ACA GCA GACGCG TTA - (811); (902)  - CTG AAC CTA GGG CGA - (916); (1345) - GAA TTCGCG ACC AGT - (1359); and (1435) - GAA TCA GAT TCT GGA - (1449).


128. The method of claim 116 wherein the promoter is a modified ompCpromoter phenotypically characterized in that said promoter exhibitshigher rates of osmotically regulated expression in relation to acorresponding ompC promoter without such point mutations.
 129. Themethod of claim 116 wherein the modified ompC promoter is without aBglII site.
 130. The method of claim 116 wherein the modified ompCpromoter comprises a mutated BglII site.
 131. The method of claim 116wherein the modified ompC promoter comprises the following sequence 5′to 3′ beginning with the mutated BglII site and ending with the ATGstart codon: AGATCX¹X²TAAX³CATCCACAGGAGGATATCTGATG, wherein: (a) X¹ isselected from the group consisting of G, C and A; (b) X² is an inserthaving from 1 to 5 nucleotides; (c) X³ is selected from the groupconsisting of A, T, G and C.
 132. A DNA comprising a modifed ompCpromoter, phenotypically characterized in that said promoter exhibitshigher rates of osmotically regulated expression in relation to acorresponding non-mutated ompC promoter.
 133. The DNA of claim 132wherein the modified ompC promoter comprises a mutated BglII site. 134.The DNA of claim 133 wherein the modified ompC promoter comprises thefollowing sequence 5′ to 3′ beginning with the mutated BglII site andending with the ATG start codon: AGATCX¹X²TAAX³CATCCACAGGAGGATATCTGATG,wherein: (a) X¹ is selected from the group consisting of G, C and A; (b)X² is an insert having from 1 to 5 nucleotides; (c) X³ is selected fromthe group consisting of A, T, G and C.
 135. The DNA of claim 134 whereinX¹ is G.
 136. The DNA of claim 134 wherein X² has from 1 to 4nucleotides.
 137. The DNA of claim 134 wherein X² has 4 nucleotides.138. The DNA of claim 134 wherein X² has 4 nucleotides, independentlyselected from the group consisting of A, T and C.
 139. The DNA of claim134 wherein X² comprises a nucleotide or nucleotide sequence selectedfrom the group consisting of ATCT; ATC; AT; TCT; CT; TC; A; T; C; and T.140. The DNA of claim 134 wherein X² is selected from the groupconsisting of ATCT; ATC; AT; TCT; CT; TC; A; T; C; and T.
 141. The DNAof claim 134 wherein X² is ATCT.
 142. The DNA of claim 134 wherein X³ isA.
 143. The DNA of claim 133 wherein the mutated BglII site of the ompCpromoter comprises the sequence: AGATCG.
 144. The DNA of claim 133wherein the mutated BglII site of the ompC promoter consists of thesequence: AGATCG.
 145. The DNA of claim 133 wherein the modified ompCpromoter comprises the following sequence 5′ to 3′ between the mutatedBglII site and the ATG start codon: AGATCTTAAACATCCACAGGAGGATATCTGATG.146. The DNA of claim 132 further comprising a plasmid maintenancesystem comprising: (a) at least one post-segregational killing function;and (b) at least one partitioning function.
 147. An expression plasmidcomprising the DNA of claim
 146. 148. The DNA of claim 132 furthercomprising a nucleotide sequence encoding a peptide or protein, theexpression of which is controlled by said modified promoter.
 149. TheDNA of claim 147, wherein the peptide or protein is selected from thegroup consisting of: heterologous antigens and green fluorescentprotein.
 150. The DNA of claim 147, wherein the peptide or protein isselected from the group consisting of: detoxified Shiga toxins.
 151. TheDNA of claim 147, wherein the peptide or protein is selected from thegroup consisting of: Shiga toxin 2 B subunit pentamers and a detoxifiedStx
 2. 152. The DNA of claim 132 further comprising a nucleotidesequence encoding a selectable marker, which marker does not conferresistance to any antibiotic which is ordinarily used in the treatmentof humans.
 153. An expression plasmid comprising the DNA of claim 152.154. The DNA of claim 132 further comprising an origin of replicationand a transcription terminator sequence in a 5′ position in relation tothe origin of replication such that transcription of the origin ofreplication is less perturbed relative to perturbance in the absence ofsuch transcriptional terminator sequence.
 155. An expression plasmidcomprising the DNA of any of claim 132.